Publications by authors named "Hay-Oak Park"

26 Publications

  • Page 1 of 1

Regulation of Cdc42 for polarized growth in budding yeast.

Microb Cell 2020 May 19;7(7):175-189. Epub 2020 May 19.

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.

The Rho GTPase Cdc42 is a central regulator of cell polarity in diverse cell types. The activity of Cdc42 is dynamically controlled in time and space to enable distinct polarization events, which generally occur along a single axis in response to spatial cues. Our understanding of the mechanisms underlying Cdc42 polarization has benefited largely from studies of the budding yeast , a genetically tractable model organism. In budding yeast, Cdc42 activation occurs in two temporal steps in the G1 phase of the cell cycle to establish a proper growth site. Here, we review findings in budding yeast that reveal an intricate crosstalk among polarity proteins for biphasic Cdc42 regulation. The first step of Cdc42 activation may determine the axis of cell polarity, while the second step ensures robust Cdc42 polarization for growth. Biphasic Cdc42 polarization is likely to ensure the proper timing of events including the assembly and recognition of spatial landmarks and stepwise assembly of a new ring of septins, cytoskeletal GTP-binding proteins, at the incipient bud site. Biphasic activation of GTPases has also been observed in mammalian cells, suggesting that biphasic activation could be a general mechanism for signal-responsive cell polarization. Cdc42 activity is necessary for polarity establishment during normal cell division and development, but its activity has also been implicated in the promotion of aging. We also discuss negative polarity signaling and emerging concepts of Cdc42 signaling in cellular aging.
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http://dx.doi.org/10.15698/mic2020.07.722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328677PMC
May 2020

Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1.

Mol Biol Cell 2019 09 14;30(20):2543-2557. Epub 2019 Aug 14.

Molecular Cellular Developmental Biology Program, The Ohio State University, Columbus, OH 43210.

The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.
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http://dx.doi.org/10.1091/mbc.E19-02-0106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6740199PMC
September 2019

Genome-Wide Studies of Rho5-Interacting Proteins That Are Involved in Oxidant-Induced Cell Death in Budding Yeast.

G3 (Bethesda) 2019 03 7;9(3):921-931. Epub 2019 Mar 7.

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210

Rho GTPases play critical roles in cell proliferation and cell death in many species. As in animal cells, cells of the budding yeast undergo regulated cell death under various physiological conditions and upon exposure to external stress. The Rho5 GTPase is necessary for oxidant-induced cell death, and cells expressing a constitutively active GTP-locked Rho5 are hypersensitive to oxidants. Yet how Rho5 regulates yeast cell death has been poorly understood. To identify genes that are involved in the Rho5-mediated cell death program, we performed two complementary genome-wide screens: one screen for oxidant-resistant deletion mutants and another screen for Rho5-associated proteins. Functional enrichment and interaction network analysis revealed enrichment for genes in pathways related to metabolism, transport, and plasma membrane organization. In particular, we find that , which is known to be involved in the CVT (Cytoplasm-to-Vacuole Targeting) pathway and mitophagy, is necessary for cell death induced by oxidants. Cells lacking Atg21 exhibit little cell death upon exposure to oxidants even when the GTP-locked Rho5 is expressed. Moreover, Atg21 interacts with Rho5 preferentially in its GTP-bound state, suggesting that Atg21 is a downstream target of Rho5 in oxidant-induced cell death. Given the high degree of conservation of Rho GTPases and autophagy from yeast to human, this study may provide insight into regulated cell death in eukaryotes in general.
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http://dx.doi.org/10.1534/g3.118.200887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404601PMC
March 2019

The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization.

Mol Biol Cell 2018 10 9;29(20):2359-2369. Epub 2018 Aug 9.

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.

The Cdc42 GTPase plays a central role in polarity development in many species. In budding yeast, Cdc42 is essential for polarized growth at the proper site and also for spontaneous cell polarization in the absence of spatial cues. Cdc42 polarization is critical for multiple events in the G1 phase prior to bud emergence, including bud-site assembly, polarization of the actin cytoskeleton, and septin filament assembly to form a ring at the new bud site. Yet the mechanism by which Cdc42 polarizes is not fully understood. Here we report that biphasic Cdc42 polarization in the G1 phase is coupled to stepwise assembly of the septin ring for bud emergence. We show that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two related Cdc42 effectors, in the first phase of Cdc42 polarization in haploid cells. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel-one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.
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http://dx.doi.org/10.1091/mbc.E18-02-0145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233053PMC
October 2018

Guidelines and recommendations on yeast cell death nomenclature.

Microb Cell 2018 Jan 1;5(1):4-31. Epub 2018 Jan 1.

Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Bari, Italy.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.
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http://dx.doi.org/10.15698/mic2018.01.607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772036PMC
January 2018

Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein.

Mol Biol Cell 2017 Dec 26;28(26):3773-3788. Epub 2017 Oct 26.

Molecular Cellular Developmental Biology Program and

In yeast and animal cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate cell polarization. In budding yeast, selection of a bud site directs polarity establishment and subsequently determines the plane of cell division. Rga1, a Cdc42 GTPase-activating protein, prevents budding within the division site by inhibiting Cdc42 repolarization. A protein complex including Nba1 and Nis1 is involved in preventing rebudding at old division sites, yet how these proteins and Rga1 might function in negative polarity signaling has been elusive. Here we show that Rga1 transiently localizes to the immediately preceding and older division sites by interacting with Nba1 and Nis1. The LIM domains of Rga1 are necessary for its interaction with Nba1, and loss of this interaction results in premature delocalization of Rga1 from the immediately preceding division site and, consequently, abnormal bud-site selection in daughter cells. However, such defects are minor in mother cells of these mutants, likely because the G1 phase is shorter and a new bud site is established prior to delocalization of Rga1. Indeed, our biphasic mathematical model of Cdc42 polarization predicts that premature delocalization of Rga1 leads to more frequent Cdc42 repolarization within the division site when the first temporal step in G1 is assumed to last longer. Spatial distribution of a Cdc42 GAP in coordination with G1 progression may thus be critical for fine-tuning the orientation of the polarity axis in yeast.
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http://dx.doi.org/10.1091/mbc.E17-01-0074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739294PMC
December 2017

Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor.

Methods Enzymol 2017 20;589:171-190. Epub 2017 Feb 20.

The Ohio State University, Columbus, OH, United States. Electronic address:

Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.
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http://dx.doi.org/10.1016/bs.mie.2017.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367485PMC
August 2017

Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast.

J Cell Sci 2015 Jun 23;128(11):2106-17. Epub 2015 Apr 23.

Molecular Cellular Developmental Biology Program, The Ohio State University, Columbus, OH 43210, USA Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.

Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42-GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on Rsr1 and its GTPase-activating protein (GAP) Bud2. Interestingly, Rga1, a Cdc42 GAP, exhibits transient localization to a site near the bud neck and to the division site during cytokinesis and G1, and this temporal change of Rga1 distribution is necessary for determination of a proper growth site. Mathematical modeling suggests that a proper axis of Cdc42 polarization in haploid cells might be established through a biphasic mechanism involving sequential positive feedback and transient negative feedback.
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http://dx.doi.org/10.1242/jcs.166538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457026PMC
June 2015

Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies.

J Mol Biol 2015 Jun 12;427(11):2039-2055. Epub 2015 Mar 12.

Molecular Cellular Developmental Biology Program, Ohio State University, OH, USA.

Complex protein networks are involved in nearly all cellular processes. To uncover these vast networks of protein interactions, various high-throughput screening technologies have been developed. Over the last decade, bimolecular fluorescence complementation (BiFC) assay has been widely used to detect protein-protein interactions (PPIs) in living cells. This technique is based on the reconstitution of a fluorescent protein in vivo. Easy quantification of the BiFC signals allows effective cell-based high-throughput screenings for protein binding partners and drugs that modulate PPIs. Recently, with the development of large screening libraries, BiFC has been effectively applied for genome-wide PPI studies and has uncovered novel protein interactions, providing new insight into protein functions. In this review, we describe the development of reagents and methods used for BiFC-based screens in yeast, plants, and mammalian cells. We also discuss the advantages and drawbacks of these methods and highlight the application of BiFC in large-scale studies.
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http://dx.doi.org/10.1016/j.jmb.2015.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417415PMC
June 2015

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae.

J Mol Biol 2015 Jun 30;427(11):2088-103. Epub 2015 Jan 30.

Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada; Department of Biochemistry, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address:

Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein-protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.
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http://dx.doi.org/10.1016/j.jmb.2015.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331858PMC
June 2015

Mathematical analysis of spontaneous emergence of cell polarity.

Bull Math Biol 2014 Aug 15;76(8):1835-65. Epub 2014 Jul 15.

Mathematical Biosciences Institute, The Ohio State University, Columbus, OH , 43210, USA,

Cell polarization, in which intracellular substances are asymmetrically distributed, enables cells to carry out specialized functions. While cell polarity is often induced by intracellular or extracellular spatial cues, spontaneous polarization (the so-called symmetry breaking) may also occur in the absence of spatial cues. Many computational models have been used to investigate the mechanisms of symmetry breaking, and it was proved that spontaneous polarization occurs when the lateral diffusion of inactive signaling molecules is much faster than that of active signaling molecules. This conclusion leaves an important question of how, as observed in many biological systems, cell polarity emerges when active and inactive membrane-bound molecules diffuse at similar rates while cycling between cytoplasm and membrane takes place. The recent studies of Rätz and Röger showed that, when the cytosolic and membrane diffusion are very different, spontaneous polarization is possible even if the membrane-bound species diffuse at the same rate. In this paper, we formulate a two-equation non-local reaction-diffusion model with general forms of positive feedback. We apply Turing stability analysis to identify parameter conditions for achieving cell polarization. Our results show that spontaneous polarization can be achieved within some parameter ranges even when active and inactive signaling molecules diffuse at similar rates. In addition, different forms of positive feedback are explored to show that a non-local molecule-mediated feedback is important for sharping the localization as well as giving rise to fast dynamics to achieve robust polarization.
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http://dx.doi.org/10.1007/s11538-014-9982-2DOI Listing
August 2014

Bud3 activates Cdc42 to establish a proper growth site in budding yeast.

J Cell Biol 2014 Jul;206(1):19-28

Department of Molecular Genetics and Molecular Cellular Developmental Biology Program, The Ohio State University, Columbus, OH 43210Department of Molecular Genetics and Molecular Cellular Developmental Biology Program, The Ohio State University, Columbus, OH 43210

Cell polarization occurs along a single axis that is generally determined by a spatial cue, yet the underlying mechanism is poorly understood. Using biochemical assays and live-cell imaging, we show that cell polarization to a proper growth site requires activation of Cdc42 by Bud3 in haploid budding yeast. Bud3 catalyzes the release of guanosine diphosphate (GDP) from Cdc42 and elevates intracellular Cdc42-guanosine triphosphate (GTP) levels in cells with inactive Cdc24, which has as of yet been the sole GDP-GTP exchange factor for Cdc42. Cdc42 is activated in two temporal steps in the G1 phase: the first depends on Bud3, whereas subsequent activation depends on Cdc24. Mutational analyses suggest that biphasic activation of Cdc42 in G1 is necessary for assembly of a proper bud site. Biphasic activation of Cdc42 or Rac GTPases may be a general mechanism for spatial cue-directed cell polarization in eukaryotes.
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http://dx.doi.org/10.1083/jcb.201402040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085707PMC
July 2014

Mapping the functional yeast ABC transporter interactome.

Nat Chem Biol 2013 Sep 7;9(9):565-72. Epub 2013 Jul 7.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.
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http://dx.doi.org/10.1038/nchembio.1293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835492PMC
September 2013

Polarization of diploid daughter cells directed by spatial cues and GTP hydrolysis of Cdc42 budding yeast.

PLoS One 2013 20;8(2):e56665. Epub 2013 Feb 20.

Mathematical Biosciences Institute, The Ohio State University, Columbus, Ohio, USA.

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056665PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577668PMC
September 2013

Coupling of septins to the axial landmark by Bud4 in budding yeast.

J Cell Sci 2013 Mar 23;126(Pt 5):1218-26. Epub 2013 Jan 23.

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.

Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern that depends on their cell type. Haploid a and α cells bud in the axial budding pattern, which requires assembly of a landmark that includes the Bud4 protein. To understand how an axial bud site is established, we performed a structure-function analysis of Bud4. Bud4 contains DUF1709 (domain of unknown function), which is similar to a part of the anillin-homology domain, and a putative Pleckstrin homology (PH) domain near to its C terminus. Although its localization depends on septins, a conserved family of GTP-binding proteins, Bud4 is necessary for the stable inheritance of septin rings during cell division. Although some anillins interact directly with septins, we find that neither DUF1709 nor the PH domain is necessary for targeting Bud4 to the mother-bud neck. Instead, this C-terminal region is crucial for association of Bud4 with Bud3 and other components of the axial landmark. Remarkably, septins colocalize with Bud4 mutant proteins that lack these C-terminal domains, forming an arc or a single ring instead of a double ring during and after cytokinesis. Interestingly, overexpression of Bud4 also induces formation of extra Bud4 rings and arcs that are associated with septins. Analyses of a series of bud4 truncation mutants suggest that at least two domains in the central region play a redundant role in targeting Bud4 to the mother-bud neck and are thus likely to interact with septins. Taken together, these results indicate that Bud4 functions as a platform that links septins to the axial landmark.
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http://dx.doi.org/10.1242/jcs.118521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3635463PMC
March 2013

Cell polarization and cytokinesis in budding yeast.

Genetics 2012 Jun;191(2):347-87

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, USA.

Asymmetric cell division, which includes cell polarization and cytokinesis, is essential for generating cell diversity during development. The budding yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, and has thus served as an attractive model for unraveling the general principles of eukaryotic cell polarization and cytokinesis. Polarity development requires G-protein signaling, cytoskeletal polarization, and exocytosis, whereas cytokinesis requires concerted actions of a contractile actomyosin ring and targeted membrane deposition. In this chapter, we discuss the mechanics and spatial control of polarity development and cytokinesis, emphasizing the key concepts, mechanisms, and emerging questions in the field.
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http://dx.doi.org/10.1534/genetics.111.132886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3374305PMC
June 2012

A novel role for the GTPase-activating protein Bud2 in the spindle position checkpoint.

PLoS One 2012 25;7(4):e36127. Epub 2012 Apr 25.

Department of Cell Biology and Physiology, Washington University, Saint Louis, Missouri, USA.

The spindle position checkpoint (SPC) ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP), and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF) for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2(R682A) to function in the checkpoint. Based on assays of heterozygous diploids, bud2(R682A), was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN). Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036127PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338500PMC
September 2012

Bud4 mediates the cell-type-specific assembly of the axial landmark in budding yeast.

J Cell Sci 2012 Aug 2;125(Pt 16):3840-9. Epub 2012 May 2.

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern depending on cell type. Haploid a and α cells bud in the axial budding pattern, which depends on a transient marker and requires proteins Bud3, Bud4, Axl1 and Axl2. Here, we report that Bud4 functions as a platform that mediates the ordered assembly of the axial landmark at the division site during M and early G1 phase. Whereas Bud4 associates with Bud3 in all cell types and in the absence of Axl1 or Axl2, Bud4 interacts with Axl1 and Axl2 mainly in haploid cells and only in the presence of all other components of the landmark. Bud4 can bind to GTP or GDP, and a GTP-binding-defective Bud4 fails to interact with Axl1 in vitro. The same bud4 mutation leads to mis-localization of Axl1 and disrupts the axial budding pattern, indicating that GTP binding to Bud4 is important for its role in bud-site selection. We also show the cell-type-specific association of the axial landmark with Bud5, a GDP/GTP exchange factor for Rsr1. Despite their expression in all cell types, Bud4 and Axl2 associate with Bud5 specifically in haploid cells and in the presence of Axl1, whose expression is limited to a and α cells. Together, our findings suggest that Bud4 plays a critical role in the assembly of the axial landmark and its link to the Rsr1 GTPase module.
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http://dx.doi.org/10.1242/jcs.103697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462081PMC
August 2012

The Rho1 GTPase acts together with a vacuolar glutathione S-conjugate transporter to protect yeast cells from oxidative stress.

Genetics 2011 Aug 30;188(4):859-70. Epub 2011 May 30.

Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210-1292, USA.

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.
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http://dx.doi.org/10.1534/genetics.111.130724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176091PMC
August 2011

The Rsr1/Bud1 GTPase interacts with itself and the Cdc42 GTPase during bud-site selection and polarity establishment in budding yeast.

Mol Biol Cell 2010 Sep 29;21(17):3007-16. Epub 2010 Jun 29.

Department of Molecular Genetics and the Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.

Cell polarization occurs along a single axis that is generally determined in response to spatial cues. In budding yeast, the Rsr1 GTPase and its regulators direct the establishment of cell polarity at the proper cortical location in response to cell type-specific cues. Here we use a combination of in vivo and in vitro approaches to understand how Rsr1 polarization is established. We find that Rsr1 associates with itself in a spatially and temporally controlled manner. The homotypic interaction and localization of Rsr1 to the mother-bud neck and to the subsequent division site are dependent on its GDP-GTP exchange factor Bud5. Analyses of rsr1 mutants suggest that Bud5 recruits Rsr1 to these sites and promotes the homodimer formation. Rsr1 also exhibits heterotypic interaction with the Cdc42 GTPase in vivo. We show that the polybasic region of Rsr1 is necessary for the efficient homotypic and heterotypic interactions, selection of a proper growth site, and polarity establishment. Our findings thus suggest that dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry.
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http://dx.doi.org/10.1091/mbc.E10-03-0232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929994PMC
September 2010

The Rho5 GTPase is necessary for oxidant-induced cell death in budding yeast.

Proc Natl Acad Sci U S A 2008 Feb 23;105(5):1522-7. Epub 2008 Jan 23.

Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA.

In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H(2)O(2)). Here, we report that the Rho5 GTPase of budding yeast is necessary for H(2)O(2)-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Delta) exhibits little cell death, whereas the constitutively active rho5(G12V) mutant exhibits excess ROS accumulation and increased cell death upon H(2)O(2) treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H(2)O(2) but also in the vacuolar lumen after H(2)O(2) treatment. Trr1 levels are elevated in rho5Delta cells but are elevated only slightly in WT and not in the rho5(G12V) cells after H(2)O(2) treatment. Taken together, these data suggest that Rho5 mediates H(2)O(2)-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.
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http://dx.doi.org/10.1073/pnas.0707359105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234177PMC
February 2008

Central roles of small GTPases in the development of cell polarity in yeast and beyond.

Microbiol Mol Biol Rev 2007 Mar;71(1):48-96

Department of Molecular Genetics, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA.

Summary: The establishment of cell polarity is critical for the development of many organisms and for the function of many cell types. A large number of studies of diverse organisms from yeast to humans indicate that the conserved, small-molecular-weight GTPases function as key signaling proteins involved in cell polarization. The budding yeast Saccharomyces cerevisiae is a particularly attractive model because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of S. cerevisiae undergo polarized growth during various phases of their life cycle, such as during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon deprivation of nutrition such as nitrogen. Substantial progress has been made in deciphering the molecular basis of cell polarity in budding yeast. In particular, it becomes increasingly clear how small GTPases regulate polarized cytoskeletal organization, cell wall assembly, and exocytosis at the molecular level and how these GTPases are regulated. In this review, we discuss the key signaling pathways that regulate cell polarization during the mitotic cell cycle and during mating.
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http://dx.doi.org/10.1128/MMBR.00028-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1847380PMC
March 2007

Interactions among Rax1p, Rax2p, Bud8p, and Bud9p in marking cortical sites for bipolar bud-site selection in yeast.

Mol Biol Cell 2004 Nov 8;15(11):5145-57. Epub 2004 Sep 8.

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210-1292, USA.

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.
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http://dx.doi.org/10.1091/mbc.e04-07-0600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC524791PMC
November 2004

Specific residues of the GDP/GTP exchange factor Bud5p are involved in establishment of the cell type-specific budding pattern in yeast.

J Biol Chem 2004 Jul 10;279(27):27980-5. Epub 2004 May 10.

Department of Molecular Genetics, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA.

Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.
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http://dx.doi.org/10.1074/jbc.C400175200DOI Listing
July 2004

Interaction between a Ras and a Rho GTPase couples selection of a growth site to the development of cell polarity in yeast.

Mol Biol Cell 2003 Dec 5;14(12):4958-70. Epub 2003 Sep 5.

Departments of Biology and Cell Biology, University of Virginia, Charlottesville, Virginia 22904, USA.

Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.
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http://dx.doi.org/10.1091/mbc.e03-06-0426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC284798PMC
December 2003

Localization of the Rsr1/Bud1 GTPase involved in selection of a proper growth site in yeast.

J Biol Chem 2002 Jul 10;277(30):26721-4. Epub 2002 Jun 10.

Graduate Program in Molecular, Cellular, and Developmental Biology, Department of Molecular Genetics, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA.

Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.
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http://dx.doi.org/10.1074/jbc.C200245200DOI Listing
July 2002
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