Limits...
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.

Show MeSH

Related in: MedlinePlus

Rho1p, Gpd1p, and Emp24p localize to peroxisomes. Double labeling fluorescence confocal microscopy of yeast cells synthesizing the indicated GFP fusions and containing a plasmid coding for peroxisomal thiolase tagged with RFP (Pot1p-RFP). The GFP chimera of Pox1p (acyl-CoA oxidase) is shown as a control. GFP chimeras of Rho1p and Gpd1p showed punctate signals colocalizing with peroxisomes. The Erg1p-GFP chimera revealed a close association between peroxisomes and lipid bodies (arrowheads; inset is a higher magnification). Emp24p-GFP colocalized with small, Pot1p-RFP–labeled peroxisomes (arrows; insets are higher magnification and longer exposure). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172632&req=5

fig4: Rho1p, Gpd1p, and Emp24p localize to peroxisomes. Double labeling fluorescence confocal microscopy of yeast cells synthesizing the indicated GFP fusions and containing a plasmid coding for peroxisomal thiolase tagged with RFP (Pot1p-RFP). The GFP chimera of Pox1p (acyl-CoA oxidase) is shown as a control. GFP chimeras of Rho1p and Gpd1p showed punctate signals colocalizing with peroxisomes. The Erg1p-GFP chimera revealed a close association between peroxisomes and lipid bodies (arrowheads; inset is a higher magnification). Emp24p-GFP colocalized with small, Pot1p-RFP–labeled peroxisomes (arrows; insets are higher magnification and longer exposure). Bar, 10 μm.

Mentions: To assess further the subcellular distribution of these candidates, each candidate was tagged at its COOH terminus with GFP by genomic integration and examined by double labeling confocal microscopy to determine its localization relative to a fluorescent peroxisomal marker, peroxisomal thiolase (Pot1p) tagged at its COOH terminus with monomeric DsRed (RFP; Pot1p-RFP). Although we were unable to detect Spf1p-GFP or Ybr159p-GFP chimeras, the other candidates revealed distributions consistent with their subcellular fractionation behavior; each was present in nonperoxisomal structures but also colocalized with peroxisomes (Fig. 4). This was most evident for Gpd1p, the punctate signal of which colocalized exclusively with the peroxisomal marker Pot1p-RFP. Gpd1p is considered primarily as a cytosolic protein that functions to shuttle electrons from cytosolically generated NADH to the mitochondrial electron transport chain through the glycerol phosphate shuttle, regenerating NAD+ in the process (Larsson et al., 1993). Its localization to peroxisomes raises the possibility that Gpd1p plays a similar role in peroxisomes, recycling NADH produced during peroxisomal β-oxidation of fatty acids (Hiltunen et al., 2003).


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, Gpd1p, and Emp24p localize to peroxisomes. Double labeling fluorescence confocal microscopy of yeast cells synthesizing the indicated GFP fusions and containing a plasmid coding for peroxisomal thiolase tagged with RFP (Pot1p-RFP). The GFP chimera of Pox1p (acyl-CoA oxidase) is shown as a control. GFP chimeras of Rho1p and Gpd1p showed punctate signals colocalizing with peroxisomes. The Erg1p-GFP chimera revealed a close association between peroxisomes and lipid bodies (arrowheads; inset is a higher magnification). Emp24p-GFP colocalized with small, Pot1p-RFP–labeled peroxisomes (arrows; insets are higher magnification and longer exposure). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Rho1p, Gpd1p, and Emp24p localize to peroxisomes. Double labeling fluorescence confocal microscopy of yeast cells synthesizing the indicated GFP fusions and containing a plasmid coding for peroxisomal thiolase tagged with RFP (Pot1p-RFP). The GFP chimera of Pox1p (acyl-CoA oxidase) is shown as a control. GFP chimeras of Rho1p and Gpd1p showed punctate signals colocalizing with peroxisomes. The Erg1p-GFP chimera revealed a close association between peroxisomes and lipid bodies (arrowheads; inset is a higher magnification). Emp24p-GFP colocalized with small, Pot1p-RFP–labeled peroxisomes (arrows; insets are higher magnification and longer exposure). Bar, 10 μm.
Mentions: To assess further the subcellular distribution of these candidates, each candidate was tagged at its COOH terminus with GFP by genomic integration and examined by double labeling confocal microscopy to determine its localization relative to a fluorescent peroxisomal marker, peroxisomal thiolase (Pot1p) tagged at its COOH terminus with monomeric DsRed (RFP; Pot1p-RFP). Although we were unable to detect Spf1p-GFP or Ybr159p-GFP chimeras, the other candidates revealed distributions consistent with their subcellular fractionation behavior; each was present in nonperoxisomal structures but also colocalized with peroxisomes (Fig. 4). This was most evident for Gpd1p, the punctate signal of which colocalized exclusively with the peroxisomal marker Pot1p-RFP. Gpd1p is considered primarily as a cytosolic protein that functions to shuttle electrons from cytosolically generated NADH to the mitochondrial electron transport chain through the glycerol phosphate shuttle, regenerating NAD+ in the process (Larsson et al., 1993). Its localization to peroxisomes raises the possibility that Gpd1p plays a similar role in peroxisomes, recycling NADH produced during peroxisomal β-oxidation of fatty acids (Hiltunen et al., 2003).

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