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. 2001 Aug 6;154(3):549-71.
doi: 10.1083/jcb.200104057.

A protein interaction map for cell polarity development

B L Drees  1 , B Sundin, E Brazeau, J P Caviston, G C Chen, W Guo, K G Kozminski, M W Lau, J J Moskow, A Tong, L R Schenkman, A McKenzie 3rd, P Brennwald, M Longtine, E Bi, C Chan, P Novick, C Boone, J R Pringle, T N Davis, S Fields, D G Drubin
Affiliations

A protein interaction map for cell polarity development

B L Drees et al. J Cell Biol. .

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.

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Figures

Figure 1.
Figure 1.
Schematic overview of connections between processes involved in cell polarity development. Major processes are color coded in this and the following figures: blue, Cdc42-signaling pathways; purple, Rho1-signaling pathways; green, septin organization; red, actin organization and endocytosis; yellow, exocytosis; brown, cell wall synthesis; turquoise, cytokinesis. Only individual proteins that appear to be branchpoints or major nodal connections between different processes are depicted. Bem4, for example, shows interactions with both Rho1 and Cdc42 GTPase pathways and with the septins. Zds1 and Zds2 link Rho1 with Cdc42 effectors and downstream processes. Ygr221c also shows interactions with both Cdc42 and Rho1 pathways. Apg17 shows interactions with proteins involved in cytokinesis, exocytosis, and Rho1 function.
Figure 2.
Figure 2.
Interaction map for proteins involved in Cdc42- and Rho1-regulated processes and in other cell polarity development pathways. Proteins that regulate Cdc42 function or that transduce signals from activated Cdc42 are shown in blue. Rho1 and its effectors are shown in purple. Cdc42-regulated pathways show interactions with proteins involved in septin organization via interactions with Bem4; with Rho1 via interactions with Zds2 and Ygr221c; and with proteins involved in cell cycle control, endocytosis, and polarized exocytosis. Cdc42 effector proteins show interactions with proteins involved in cytokinesis, microtubule stability, polarized growth, actin assembly, polarized secretion, and cell wall synthesis. Proteins involved in septin organization may interact with Rho1 via Bem4. Rho1 involvement in nuclear migration, actin/myosin ring contraction, and septum formation (exocytosis) during cytokinesis is suggested by Apg17-mediated connections between proteins involved in these processes. Other interactions suggest connections between late exocytic and early endocytic processes, between early and late steps in secretory pathways, and between exocytosis and autophagy.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
Fluorescence micrographs of proteins tagged at the COOH terminus with YFP (A–N). YFP signal is shown in green. Cells were outlined by staining with Alexa fluor 633 conjugated to concanavalin A (blue). (A and B) Ykl082c-YFP; (C and D) Ycr086w-YFP; (E and F) Ygr221c-YFP; (G and H) Yil079c/Air1-YFP; (I and J) Ylr423c/Apg17-YFP; (K and L) Ypr171w-YFP; (M and N) Yor284w-YFP. (O and P) Immunofluorescence micrographs of Yhr149c tagged with a 13Myc epitope. GFP-tagged Yhr149 exhibits the same localization although the GFP signal is extremely weak. Bar, 5 μm.
Figure 5.
Figure 5.
Fluorescence micrographs of Ypr171w-YFP showing localization to actin cortical patches. Cells were outlined by staining with Alexa fluor 633 conjugated to concanavalin A (blue). (A and D) Ypr171w-YFP (green); (B and E) Abp1-CFP (red); (C and F) merged image. (G and H) Ypr171w-YFP (G) and Abp1-CFP (H) in an ark1 prk1 deletion strain. Bar, 5 μm.
Figure 6.
Figure 6.
Fluorescence micrographs of Yor284w-YFP showing the effect of actin depolarization (A–D), localization to the spindle pole body (E–J), and localization relative to the actin cytoskeleton (K–P). Cells were outlined by staining with Alexa fluor 633 conjugated to concanavalin A (blue). (A and B) Yor284w-YFP; (C and D) Yor284w-YFP in an ark1 prk1 deletion strain. (E and H) Yor284w-YFP (green); (F and I) Spc29-CFP (red); (G and J) merged image. (K and N) Yor284w-YFP (green); (L and O) Abp1-CFP (red); (M and P) merged image. Bar, 5 μm.
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