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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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For uncharacterized proteins identified in the screen, some insights into their roles in polarized growth can be obtained from their functional associations in gene networks. The gene neighbors (yellow circles) of five uncharacterized proteins (red circles) in a functional gene network(17) are plotted, indicating functional linkages by blue lines. Black circles enclose proteins already known to be part of the shmoo tip network. Panels A and B show two such uncharacterized sets of proteins that can be functionally tied via the network to different aspects of the shmoo tip gene network. In (A), Yor304C-a is functionally connected to BUD6 on the basis of evidence from affinity purification/mass spectrometry experiments summarized in the network. In (B), RVS161, CAP1 and CAP2, components of the known shmoo tip network, also connect to the uncharacterized genes, Yer071c and Ycr043c via protein complex data acquired by mass spectrometry.(27)
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fig7: For uncharacterized proteins identified in the screen, some insights into their roles in polarized growth can be obtained from their functional associations in gene networks. The gene neighbors (yellow circles) of five uncharacterized proteins (red circles) in a functional gene network(17) are plotted, indicating functional linkages by blue lines. Black circles enclose proteins already known to be part of the shmoo tip network. Panels A and B show two such uncharacterized sets of proteins that can be functionally tied via the network to different aspects of the shmoo tip gene network. In (A), Yor304C-a is functionally connected to BUD6 on the basis of evidence from affinity purification/mass spectrometry experiments summarized in the network. In (B), RVS161, CAP1 and CAP2, components of the known shmoo tip network, also connect to the uncharacterized genes, Yer071c and Ycr043c via protein complex data acquired by mass spectrometry.(27)

Mentions: Several uncharacterized proteins recovered in our screen, such as Ymr295c,(49) Ydr348c, and Yor304c-a, localize to both the bud(9) and shmoo tips suggesting a role in the general polarization machinery. Examination of these proteins’ functional associations in the yeast functional gene network(27) provides some suggestions for their connection to particular aspects of polarized growth (Figure 7A): Yor304c-a is most tightly functionally associated with Bud6, a central protein in signaling polarization (also recovered in the screen) and Duo1, a cytoskeletal protein. Ymr295c and Ydr348c link to each other, with the former also strongly associated with glycolytic transcription factor Gcr1, and the latter associated with cell cycle progression genes Clb2 and Cdc28. This may suggest a role for these genes connecting polarization with processes such as cell cycle state and metabolism.


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)

For uncharacterized proteins identified in the screen, some insights into their roles in polarized growth can be obtained from their functional associations in gene networks. The gene neighbors (yellow circles) of five uncharacterized proteins (red circles) in a functional gene network(17) are plotted, indicating functional linkages by blue lines. Black circles enclose proteins already known to be part of the shmoo tip network. Panels A and B show two such uncharacterized sets of proteins that can be functionally tied via the network to different aspects of the shmoo tip gene network. In (A), Yor304C-a is functionally connected to BUD6 on the basis of evidence from affinity purification/mass spectrometry experiments summarized in the network. In (B), RVS161, CAP1 and CAP2, components of the known shmoo tip network, also connect to the uncharacterized genes, Yer071c and Ycr043c via protein complex data acquired by mass spectrometry.(27)
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig7: For uncharacterized proteins identified in the screen, some insights into their roles in polarized growth can be obtained from their functional associations in gene networks. The gene neighbors (yellow circles) of five uncharacterized proteins (red circles) in a functional gene network(17) are plotted, indicating functional linkages by blue lines. Black circles enclose proteins already known to be part of the shmoo tip network. Panels A and B show two such uncharacterized sets of proteins that can be functionally tied via the network to different aspects of the shmoo tip gene network. In (A), Yor304C-a is functionally connected to BUD6 on the basis of evidence from affinity purification/mass spectrometry experiments summarized in the network. In (B), RVS161, CAP1 and CAP2, components of the known shmoo tip network, also connect to the uncharacterized genes, Yer071c and Ycr043c via protein complex data acquired by mass spectrometry.(27)
Mentions: Several uncharacterized proteins recovered in our screen, such as Ymr295c,(49) Ydr348c, and Yor304c-a, localize to both the bud(9) and shmoo tips suggesting a role in the general polarization machinery. Examination of these proteins’ functional associations in the yeast functional gene network(27) provides some suggestions for their connection to particular aspects of polarized growth (Figure 7A): Yor304c-a is most tightly functionally associated with Bud6, a central protein in signaling polarization (also recovered in the screen) and Duo1, a cytoskeletal protein. Ymr295c and Ydr348c link to each other, with the former also strongly associated with glycolytic transcription factor Gcr1, and the latter associated with cell cycle progression genes Clb2 and Cdc28. This may suggest a role for these genes connecting polarization with processes such as cell cycle state and metabolism.

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