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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 enriches with peroxisomes. (A) Organellar 20KgP fractions from cells expressing different pA chimeras or wild-type cells were separated by isopycnic density gradient centrifugation and analyzed by Western blotting. Fractions enriched for peroxisomes (P; 8–10) were identified by the peroxisomal proteins Pex13p-pA, Fox2p, and Mls1p. Peak mitochondrial and Golgi fractions were identified by Sdh2p and Vps15p-pA, respectively. (B) The protein concentration and density profiles for each gradient fraction are presented.
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fig3: Rho1p enriches with peroxisomes. (A) Organellar 20KgP fractions from cells expressing different pA chimeras or wild-type cells were separated by isopycnic density gradient centrifugation and analyzed by Western blotting. Fractions enriched for peroxisomes (P; 8–10) were identified by the peroxisomal proteins Pex13p-pA, Fox2p, and Mls1p. Peak mitochondrial and Golgi fractions were identified by Sdh2p and Vps15p-pA, respectively. (B) The protein concentration and density profiles for each gradient fraction are presented.

Mentions: Although the growth assay can serve to implicate proteins in peroxisome function, genetic redundancy, buffering, or subtle effects can allow cells lacking bona fide peroxins to still grow on oleic acid medium (for review see Hiltunen et al., 2003). Similarly, mutations in nonperoxisomal proteins could also lead to growth defects in oleic acid medium through pleiotropic or nonspecific effects. Thus, further validation of the data set came from additional localization studies of several high-scoring candidates from Groups 1 and 3. As mentioned above, the quantitative MS approach was designed to identify peroxisomal proteins but was also expected to identify proteins that might be localized to one or more additional compartments. Therefore, proteins representative of different cellular compartments were targeted: glycerol-3-phosphate dehydrogenase, Gpd1p (cytosol); a P-type ATPase, Spf1p; a protein with 3-ketoreductase activity, Ybr159p; and a COP II coat component, Emp24p (ER); the fatty acid transporter, Faa1p; the squalene epoxidase, Erg1p; and sterol 24-C-methyltransferase, Erg6p (lipid bodies); the small GTPase, Rho1p (plasma [and endo] membranes). Yeast strains containing genomically integrated protein A fusions of each candidate were made, and the behavior of each in subcellular fractionation was assessed by Western blot analysis (Fig. 3). Although each candidate was detected in fractions of low density, a portion of each also cofractionated with peroxisomes, which were detected using the peroxisomal marker proteins Fox2p, Mls1p, and Pex13p (for review see Hiltunen et al., 2003). As controls, marker proteins for endosomes and the late Golgi (Vps15p; Herman et al., 1991), the mitochondrion (Sdh2p; Robinson and Lemire, 1996), and the nucleus (Gsp1p; Moore, 1998; unpublished data) were also investigated. These proteins were not detected in the peroxisomal fractions. These data support previous studies localizing several of these components to other membranes, but also support our MS data and suggest that a subpopulation of each protein is associated with peroxisomes isolated from oleate-induced cells.


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 enriches with peroxisomes. (A) Organellar 20KgP fractions from cells expressing different pA chimeras or wild-type cells were separated by isopycnic density gradient centrifugation and analyzed by Western blotting. Fractions enriched for peroxisomes (P; 8–10) were identified by the peroxisomal proteins Pex13p-pA, Fox2p, and Mls1p. Peak mitochondrial and Golgi fractions were identified by Sdh2p and Vps15p-pA, respectively. (B) The protein concentration and density profiles for each gradient fraction are presented.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Rho1p enriches with peroxisomes. (A) Organellar 20KgP fractions from cells expressing different pA chimeras or wild-type cells were separated by isopycnic density gradient centrifugation and analyzed by Western blotting. Fractions enriched for peroxisomes (P; 8–10) were identified by the peroxisomal proteins Pex13p-pA, Fox2p, and Mls1p. Peak mitochondrial and Golgi fractions were identified by Sdh2p and Vps15p-pA, respectively. (B) The protein concentration and density profiles for each gradient fraction are presented.
Mentions: Although the growth assay can serve to implicate proteins in peroxisome function, genetic redundancy, buffering, or subtle effects can allow cells lacking bona fide peroxins to still grow on oleic acid medium (for review see Hiltunen et al., 2003). Similarly, mutations in nonperoxisomal proteins could also lead to growth defects in oleic acid medium through pleiotropic or nonspecific effects. Thus, further validation of the data set came from additional localization studies of several high-scoring candidates from Groups 1 and 3. As mentioned above, the quantitative MS approach was designed to identify peroxisomal proteins but was also expected to identify proteins that might be localized to one or more additional compartments. Therefore, proteins representative of different cellular compartments were targeted: glycerol-3-phosphate dehydrogenase, Gpd1p (cytosol); a P-type ATPase, Spf1p; a protein with 3-ketoreductase activity, Ybr159p; and a COP II coat component, Emp24p (ER); the fatty acid transporter, Faa1p; the squalene epoxidase, Erg1p; and sterol 24-C-methyltransferase, Erg6p (lipid bodies); the small GTPase, Rho1p (plasma [and endo] membranes). Yeast strains containing genomically integrated protein A fusions of each candidate were made, and the behavior of each in subcellular fractionation was assessed by Western blot analysis (Fig. 3). Although each candidate was detected in fractions of low density, a portion of each also cofractionated with peroxisomes, which were detected using the peroxisomal marker proteins Fox2p, Mls1p, and Pex13p (for review see Hiltunen et al., 2003). As controls, marker proteins for endosomes and the late Golgi (Vps15p; Herman et al., 1991), the mitochondrion (Sdh2p; Robinson and Lemire, 1996), and the nucleus (Gsp1p; Moore, 1998; unpublished data) were also investigated. These proteins were not detected in the peroxisomal fractions. These data support previous studies localizing several of these components to other membranes, but also support our MS data and suggest that a subpopulation of each protein is associated with peroxisomes isolated from oleate-induced cells.

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