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Published online 13 December 2004. doi:10.1083/jcb.200404119
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 167, Number 6, 1099-1112
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Article

 Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane

Marcello Marelli1, Jennifer J. Smith1, Sunhee Jung1, Eugene Yi1, Alexey I. Nesvizhskii1, Rowan H. Christmas1, Ramsey A. Saleem1, Yuen Yi C. Tam2, Andrei Fagarasanu2, David R. Goodlett1, Ruedi Aebersold1, Richard A. Rachubinski2, and John D. Aitchison1,2

1 Institute for Systems Biology, Seattle, WA 98103
2 Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Correspondence to John D. Aitchison: jaitchison{at}systemsbiology.org

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.

Abbreviations used in this paper: AP, affinity-purified peroxisomal membrane; DsRed, Discosoma sp. red fluorescent protein; ICAT, isotope-coded affinity tag; MS, mass spectrometry; µLC/ESI-MS/MS, microcapillary liquid chromatography/electrospray ionization tandem MS; PTS, peroxisomal targeting signal; RFP, monomeric DsRed; SGD, Saccharomyces Genome Database.


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