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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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Schematic summarizing the spatial reorganization of organelles and protein complexes along the major polarization axis formed during shmooing. An alpha factor-induced polarization axis is established which leads to the spatial reorganization of multiple organelles in a roughly ordered fashion from proximal to the shmoo tip to the distal end of the cell. At the extreme growth tip lie proteins of exocytosis and mating, ringed by proteins associated with actin cortical patches and endocytosis, and—progressively moving away from the proximal tip—to the septins, spindle pole body, and nucleus, with the nucleolus generally oriented on the far side of the nucleus from the shmoo tip.
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fig8: Schematic summarizing the spatial reorganization of organelles and protein complexes along the major polarization axis formed during shmooing. An alpha factor-induced polarization axis is established which leads to the spatial reorganization of multiple organelles in a roughly ordered fashion from proximal to the shmoo tip to the distal end of the cell. At the extreme growth tip lie proteins of exocytosis and mating, ringed by proteins associated with actin cortical patches and endocytosis, and—progressively moving away from the proximal tip—to the septins, spindle pole body, and nucleus, with the nucleolus generally oriented on the far side of the nucleus from the shmoo tip.

Mentions: We report a large-scale imaging-based screen for proteins showing spatial localization to the growing tip of the yeast mating projection following pheromone response. A summary of our findings is presented in Figure 8. Our screen has enabled us to align the proteome down to the level of individual proteins identified along the polarization axis induced by pheromone stimulation. Upon the basis of this systematic assay, we demonstrate that the bulk of shmoo-tip localized proteins are conserved with those localized to the bud tip during vegetative growth. These data therefore support a model for the adaptive reuse of polarized growth machinery between different polarized growth processes. For example, Huh et al.(9) had shown that actin related genes are structured along the polarization axis during budding. We show that this observation is true during mating as well. This process is not automatic since the budding and mating projection sites are distinct and therefore the point of origin of cellular asymmetry is likely also different. Moreover, the 67 proteins shared between the mating and budding polarization processes (Figure 5) are significantly more conserved between human and yeast than expected by chance (39 of the 67 yeast genes have human orthologs,(56)p < 0.023, chi-square test), indicating that the functions of these core polarization components are likely to be preserved across eukaryotes. Although the process of establishment of cell polarity by proteins such as Cdc42, Cdc24 and Far1 are well-studied, the exact molecular mechanisms toward predictive cellular models are still wanting.(57) Our efforts are a first step toward screening proteome-wide dynamics to understand precisely how biological systems spatially coordinate protein interactions and regulate their dynamics.


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 summarizing the spatial reorganization of organelles and protein complexes along the major polarization axis formed during shmooing. An alpha factor-induced polarization axis is established which leads to the spatial reorganization of multiple organelles in a roughly ordered fashion from proximal to the shmoo tip to the distal end of the cell. At the extreme growth tip lie proteins of exocytosis and mating, ringed by proteins associated with actin cortical patches and endocytosis, and—progressively moving away from the proximal tip—to the septins, spindle pole body, and nucleus, with the nucleolus generally oriented on the far side of the nucleus from the shmoo tip.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig8: Schematic summarizing the spatial reorganization of organelles and protein complexes along the major polarization axis formed during shmooing. An alpha factor-induced polarization axis is established which leads to the spatial reorganization of multiple organelles in a roughly ordered fashion from proximal to the shmoo tip to the distal end of the cell. At the extreme growth tip lie proteins of exocytosis and mating, ringed by proteins associated with actin cortical patches and endocytosis, and—progressively moving away from the proximal tip—to the septins, spindle pole body, and nucleus, with the nucleolus generally oriented on the far side of the nucleus from the shmoo tip.
Mentions: We report a large-scale imaging-based screen for proteins showing spatial localization to the growing tip of the yeast mating projection following pheromone response. A summary of our findings is presented in Figure 8. Our screen has enabled us to align the proteome down to the level of individual proteins identified along the polarization axis induced by pheromone stimulation. Upon the basis of this systematic assay, we demonstrate that the bulk of shmoo-tip localized proteins are conserved with those localized to the bud tip during vegetative growth. These data therefore support a model for the adaptive reuse of polarized growth machinery between different polarized growth processes. For example, Huh et al.(9) had shown that actin related genes are structured along the polarization axis during budding. We show that this observation is true during mating as well. This process is not automatic since the budding and mating projection sites are distinct and therefore the point of origin of cellular asymmetry is likely also different. Moreover, the 67 proteins shared between the mating and budding polarization processes (Figure 5) are significantly more conserved between human and yeast than expected by chance (39 of the 67 yeast genes have human orthologs,(56)p < 0.023, chi-square test), indicating that the functions of these core polarization components are likely to be preserved across eukaryotes. Although the process of establishment of cell polarity by proteins such as Cdc42, Cdc24 and Far1 are well-studied, the exact molecular mechanisms toward predictive cellular models are still wanting.(57) Our efforts are a first step toward screening proteome-wide dynamics to understand precisely how biological systems spatially coordinate protein interactions and regulate their dynamics.

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