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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.

Narayanaswamy R, Moradi EK, Niu W, Hart GT, Davis M, McGary KL, Ellington AD, Marcotte EM - J. Proteome Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Departments of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712.

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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Related in: MedlinePlus

Schematic of the work flow for performing the proteome-wide localization screen. The GFP tagged yeast library was treated with mating pheromone, the cells were fixed, and then microarrayed onto a microscope slide. High-throughput microscopy of the arrayed strains revealed proteins localized to the polarized growth tip. This gene set was expanded by training a machine learning classifier to distinguish the observed proteins from the rest of the proteome on the basis of their vegetative expression patterns(9) and their connections in a functional gene network.(17) Additional proteins identified from the classifier were manually validated, resulting in a final set of 74 proteins localized to the polarized growth tip of the mating projection.
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fig3: Schematic of the work flow for performing the proteome-wide localization screen. The GFP tagged yeast library was treated with mating pheromone, the cells were fixed, and then microarrayed onto a microscope slide. High-throughput microscopy of the arrayed strains revealed proteins localized to the polarized growth tip. This gene set was expanded by training a machine learning classifier to distinguish the observed proteins from the rest of the proteome on the basis of their vegetative expression patterns(9) and their connections in a functional gene network.(17) Additional proteins identified from the classifier were manually validated, resulting in a final set of 74 proteins localized to the polarized growth tip of the mating projection.

Mentions: An overview of the cell microarray assay is presented in Figure 3. Briefly, after treating the library of GFP-tagged fusion protein expression strains with alpha factor, we fixed and robotically printed(9) the strains onto poly lysine-coated microscope slides(10) and imaged the fixed strains using automated microscopy. We screened for proteins showing a clear localization proximal to the shmoo tip. In all, 187 proteins were chosen from the initial screen, based on a lenient criterion that included even marginal examples. These 187 strains were then manually retested in the absence of fixative, which improved signal-to-noise value because of reduced background fluorescence. The follow-up screen yielded 37 strains in which GFP fusion proteins were consistently localized to the shmoo tip (Table 1).


Systematic definition of protein constituents along the major polarization axis reveals an adaptive reuse of the polarization machinery in pheromone-treated budding yeast.

Narayanaswamy R, Moradi EK, Niu W, Hart GT, Davis M, McGary KL, Ellington AD, Marcotte EM - J. Proteome Res. (2009)

Schematic of the work flow for performing the proteome-wide localization screen. The GFP tagged yeast library was treated with mating pheromone, the cells were fixed, and then microarrayed onto a microscope slide. High-throughput microscopy of the arrayed strains revealed proteins localized to the polarized growth tip. This gene set was expanded by training a machine learning classifier to distinguish the observed proteins from the rest of the proteome on the basis of their vegetative expression patterns(9) and their connections in a functional gene network.(17) Additional proteins identified from the classifier were manually validated, resulting in a final set of 74 proteins localized to the polarized growth tip of the mating projection.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651748&req=5

fig3: Schematic of the work flow for performing the proteome-wide localization screen. The GFP tagged yeast library was treated with mating pheromone, the cells were fixed, and then microarrayed onto a microscope slide. High-throughput microscopy of the arrayed strains revealed proteins localized to the polarized growth tip. This gene set was expanded by training a machine learning classifier to distinguish the observed proteins from the rest of the proteome on the basis of their vegetative expression patterns(9) and their connections in a functional gene network.(17) Additional proteins identified from the classifier were manually validated, resulting in a final set of 74 proteins localized to the polarized growth tip of the mating projection.
Mentions: An overview of the cell microarray assay is presented in Figure 3. Briefly, after treating the library of GFP-tagged fusion protein expression strains with alpha factor, we fixed and robotically printed(9) the strains onto poly lysine-coated microscope slides(10) and imaged the fixed strains using automated microscopy. We screened for proteins showing a clear localization proximal to the shmoo tip. In all, 187 proteins were chosen from the initial screen, based on a lenient criterion that included even marginal examples. These 187 strains were then manually retested in the absence of fixative, which improved signal-to-noise value because of reduced background fluorescence. The follow-up screen yielded 37 strains in which GFP fusion proteins were consistently localized to the shmoo tip (Table 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Departments of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712.

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.

Show MeSH
Related in: MedlinePlus