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Systematic Definition of Protein Constituents along the Major Polarization Axis Reveals an Adaptive Reuse of the Polarization Machinery in Pheromone-Treated Budding Yeast

Rammohan Narayanaswamy^†, Emily K. Moradi^†^§^#, Wei Niu^†^#, G. Traver Hart^†^#, Matthew Davis^†^#, Kriston L. McGary^†^#, Andrew D. Ellington*^†^‡ and Edward M. Marcotte*^†^‡

Center for Systems and Synthetic Biology, Departments of Chemistry and Biochemistry, and Biomedical Engineering, Institute for Cellular and Molecular Biology, 2500 Speedway, University of Texas, Austin, Texas 78712

J. Proteome Res., 2009, 8 (1), pp 6–19

DOI: 10.1021/pr800524g

Publication Date (Web): December 3, 2008

†

Center for Systems and Synthetic Biology, University of Texas.

Institute for Cellular and Molecular Biology, University of Texas.

Current address: Department of Pathology, Yale University School of Medicine, 310 Cedar Street LH 214, P.O. Box 208023, New Haven, CT 06520-8023.

Department of Biomedical Engineering, University of Texas.

These authors contributed equally.

Current address: Department of Molecular, Cellular, and Developmental Biology, KBT 926, Yale University, P.O. Box 208103, New Haven, CT 06620-8103.

Current address: QB3 Institute, 387 Stanley Hall # 3220, University of California, Berkeley, CA 94720-3220.

* To whom correspondence should be addressed. E-mail: (E.M.M.) marcotte@icmb.utexas.edu, (A.D.E.) andy.ellington@mail.utexas.edu.

‡

Department of Chemistry and Biochemistry, University of Texas.

This article is part of the

Spatial and Temporal Proteomics

special issue.

Abstract

Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the ‘shmoo’) by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.

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