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A protein interaction map for cell polarity development.

Drees BL, Sundin B, Brazeau E, Caviston JP, Chen GC, Guo W, Kozminski KG, Lau MW, Moskow JJ, Tong A, Schenkman LR, McKenzie A, Brennwald P, Longtine M, Bi E, Chan C, Novick P, Boone C, Pringle JR, Davis TN, Fields S, Drubin DG - J. Cell Biol. (2001)

Bottom Line: Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis.Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery.In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Show MeSH
Protein interactions involved in actin assembly and actin functions in endocytosis, cytokinesis, and morphogenesis. Cdc42 effectors show interactions with proteins involved in endocytosis and cytokinesis. Interactions between Ynl094w, several actin cytoskeleton proteins, and Swe1 and Hsl7 may underlie the morphogenesis checkpoint that monitors actin assembly. Several interactions between SH3 domain–containing proteins (shaded rectangles) and proteins containing proline-rich putative SH3 binding sites (shaded ovals) are shown.
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fig3: Protein interactions involved in actin assembly and actin functions in endocytosis, cytokinesis, and morphogenesis. Cdc42 effectors show interactions with proteins involved in endocytosis and cytokinesis. Interactions between Ynl094w, several actin cytoskeleton proteins, and Swe1 and Hsl7 may underlie the morphogenesis checkpoint that monitors actin assembly. Several interactions between SH3 domain–containing proteins (shaded rectangles) and proteins containing proline-rich putative SH3 binding sites (shaded ovals) are shown.

Mentions: 68 proteins with various functions in cell polarity development were used as DNA binding domain hybrids for two-hybrid screens. These included Rho-type GTPases and their regulators and effectors, actin cortical patch components, septin-associated proteins, and proteins involved in secretion (Table I). The yeast ORF-Gal4 activation domain fusion array used in our experiments expresses ∼85–90% of the predicted ORFs of S. cerevisiae (Hudson et al., 1997; Uetz et al., 2000). 14 proteins, Aip2, Bud5, Bud6, Bud7, Bud9, Cap2, Cdc3, Cdc10, Iqg1, Kin1, Msb1, Sec9, Snc1, and Snc2, showed no reproducible two-hybrid interactions when used as baits in our screens. Screens of the other 54 baits found from 1 to 13 interactions each. Overall, 196 reproducible two-hybrid positives were detected that describe 191 putative protein–protein interactions involving 110 proteins (Table I and Figs. 1–3) . 128 interactions had not been described previously and 44 involve 20 proteins of unknown function. The results of this study clearly do not represent all of the detectable or probable interactions between the proteins examined. The lack of an interaction detected in this analysis is not necessarily meaningful, as some constructs in the array might not express the expected fusion proteins or might express them in a nonfunctional form due to the Gal4 fusion. Differences in fusion construction, construct expression, strain background, and selection stringency are also factors that may account for discrepancies between the set of interactions seen here and those found in other studies.


A protein interaction map for cell polarity development.

Drees BL, Sundin B, Brazeau E, Caviston JP, Chen GC, Guo W, Kozminski KG, Lau MW, Moskow JJ, Tong A, Schenkman LR, McKenzie A, Brennwald P, Longtine M, Bi E, Chan C, Novick P, Boone C, Pringle JR, Davis TN, Fields S, Drubin DG - J. Cell Biol. (2001)

Protein interactions involved in actin assembly and actin functions in endocytosis, cytokinesis, and morphogenesis. Cdc42 effectors show interactions with proteins involved in endocytosis and cytokinesis. Interactions between Ynl094w, several actin cytoskeleton proteins, and Swe1 and Hsl7 may underlie the morphogenesis checkpoint that monitors actin assembly. Several interactions between SH3 domain–containing proteins (shaded rectangles) and proteins containing proline-rich putative SH3 binding sites (shaded ovals) are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196425&req=5

fig3: Protein interactions involved in actin assembly and actin functions in endocytosis, cytokinesis, and morphogenesis. Cdc42 effectors show interactions with proteins involved in endocytosis and cytokinesis. Interactions between Ynl094w, several actin cytoskeleton proteins, and Swe1 and Hsl7 may underlie the morphogenesis checkpoint that monitors actin assembly. Several interactions between SH3 domain–containing proteins (shaded rectangles) and proteins containing proline-rich putative SH3 binding sites (shaded ovals) are shown.
Mentions: 68 proteins with various functions in cell polarity development were used as DNA binding domain hybrids for two-hybrid screens. These included Rho-type GTPases and their regulators and effectors, actin cortical patch components, septin-associated proteins, and proteins involved in secretion (Table I). The yeast ORF-Gal4 activation domain fusion array used in our experiments expresses ∼85–90% of the predicted ORFs of S. cerevisiae (Hudson et al., 1997; Uetz et al., 2000). 14 proteins, Aip2, Bud5, Bud6, Bud7, Bud9, Cap2, Cdc3, Cdc10, Iqg1, Kin1, Msb1, Sec9, Snc1, and Snc2, showed no reproducible two-hybrid interactions when used as baits in our screens. Screens of the other 54 baits found from 1 to 13 interactions each. Overall, 196 reproducible two-hybrid positives were detected that describe 191 putative protein–protein interactions involving 110 proteins (Table I and Figs. 1–3) . 128 interactions had not been described previously and 44 involve 20 proteins of unknown function. The results of this study clearly do not represent all of the detectable or probable interactions between the proteins examined. The lack of an interaction detected in this analysis is not necessarily meaningful, as some constructs in the array might not express the expected fusion proteins or might express them in a nonfunctional form due to the Gal4 fusion. Differences in fusion construction, construct expression, strain background, and selection stringency are also factors that may account for discrepancies between the set of interactions seen here and those found in other studies.

Bottom Line: Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis.Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery.In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Show MeSH