The Rho GTPase Rho3 Has a Direct Role in Exocytosis That Is Distinct from Its Role in Actin Polarity

Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.

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Functions for Cdc42p BEM Adaptors in Regulating a Differentiation-Type MAP Kinase Pathway

10.1101/786483 ◽

2019 ◽

Author(s):

Sukanya Basu ◽

Beatriz González ◽

Boyang Li ◽

Garrett Kimble ◽

Keith G. Kozminski ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Polarity ◽

Rho Gtpase ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

List Type ◽

Activation Kinetics ◽

Closed Conformation ◽

Mapk Pathways ◽

Effector Pathways

ABSTRACTRho GTPases regulate cell polarity and signal transduction pathways to control morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not clear. To address this question, Cdc42p-dependent MAPK pathways were compared in the filamentous (∑1278b) strain background. Each MAPK pathway showed a unique activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared to the mating and HOG pathways. A previously characterized version of Cdc42p, Cdc42pE100A, that is specifically defective for fMAPK pathway signaling, was defective for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the fMAPK pathway. In the fMAPK pathway, Bem1p recruited Ste20p to the plasma membrane, cycled between an open and closed conformation, and interacted with the GEF for Cdc42, Cdc24p. Bem1p also regulated effector pathways in different ways, behaving as a multi-functional adaptor in some pathways and an inert scaffold in others. Genetic suppression tests showed that Bem4p and Bem1p regulate the fMAPK pathway in an ordered sequence. Collectively, the study demonstrates unique and sequential functions for Rho GTPase adaptors in regulating MAPK pathways.HIGHLIGHTSComparing Cdc42p-dependent MAPK pathways showed that the fMAPK pathway had slow activation kinetics compared to the mating and HOG pathways.A collection of cdc42 alleles was tested for MAPK pathway functions. § Cdc42pE100A, previously characterized as being specifically defective for fMAPK signaling, showed reduced interaction with the fMAPK pathway adaptor Bem4p.§ Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK signaling.The polarity adaptor Bem1p regulated the fMAPK pathway. § Bem1p regulated the fMAPK pathway by recruiting Ste20p to the plasma membrane, cycling between an open and closed conformation, and interacting with the Cdc42p GEF, Cdc24p.Different domains of Bem1p had different roles in regulating effector pathways. § Bem1p may function as a multi-functional adaptor in some pathways and an inert scaffold in others.Bem4p and Bem1p regulated the fMAPK pathway in an ordered sequence. § The data support a model where Bem4p recruits Cdc24p to GDP-Cdc42p, and Bem1p directs GTP-Cdc42p to Ste20p at the plasma membrane.§ The bud-site GTPase Rsr1p regulates Cdc24p in the fMAPK pathway but does not initiate signaling.

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Interaction between a Ras and a Rho GTPase Couples Selection of a Growth Site to the Development of Cell Polarity in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0426 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4958-4970 ◽

Cited By ~ 67

Author(s):

Keith G. Kozminski ◽

Laure Beven ◽

Elizabeth Angerman ◽

Amy Hin Yan Tong ◽

Charles Boone ◽

...

Keyword(s):

Cell Polarity ◽

Rho Gtpase ◽

Cell Cortex ◽

Nucleotide Exchange ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Site ◽

Ras Family ◽

Rho Family ◽

Polarity Establishment ◽

Selection Of

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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The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.120.5.1203 ◽

1993 ◽

Vol 120 (5) ◽

pp. 1203-1215 ◽

Cited By ~ 59

Author(s):

K Kuchler ◽

H G Dohlman ◽

J Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Gene Product ◽

Cell Polarity ◽

Polyclonal Antibodies ◽

Apparent Molecular Weight ◽

Subcellular Fractionation ◽

Mating Pheromone ◽

Yeast Saccharomyces Cerevisiae ◽

Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

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Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

Eukaryotic Cell ◽

10.1128/ec.00042-12 ◽

2012 ◽

Vol 11 (5) ◽

pp. 590-600 ◽

Cited By ~ 6

Author(s):

Fabien Lefèbvre ◽

Valérie Prouzet-Mauléon ◽

Michel Hugues ◽

Marc Crouzet ◽

Aurélie Vieillemard ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Cell Cycle Progression ◽

Secretory Pathway ◽

Rho Gtpase ◽

Secretory Vesicles ◽

Gtpase Activating Protein ◽

Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

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The Cooh-Terminal Domain of Myo2p, a Yeast Myosin V, Has a Direct Role in Secretory Vesicle Targeting

The Journal of Cell Biology ◽

10.1083/jcb.147.4.791 ◽

1999 ◽

Vol 147 (4) ◽

pp. 791-808 ◽

Cited By ~ 184

Author(s):

Daniel Schott ◽

Jackson Ho ◽

David Pruyne ◽

Anthony Bretscher

Keyword(s):

Secretory Vesicle ◽

Restrictive Temperature ◽

Secretory Vesicles ◽

Tail Domain ◽

Myosin V ◽

Direct Role ◽

Rab Protein ◽

Polarized Delivery ◽

Actin Cables ◽

Type V

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable–dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

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Electrostatic plasma membrane targeting contributes to Dlg function in cell polarity and tumorigenesis

Development ◽

10.1242/dev.196956 ◽

2021 ◽

pp. dev.196956

Author(s):

Juan Lu ◽

Wei Dong ◽

Yan Tao ◽

Yang Hong

Keyword(s):

Plasma Membrane ◽

Tumor Suppressor ◽

Epithelial Cells ◽

Cell Polarity ◽

Significant Role ◽

Discs Large ◽

Cortical Localization ◽

Polarity Proteins ◽

Positively Charged

Discs large (Dlg) is an essential polarity protein and a tumor suppressor originally characterized in Drosophila but is also well conserved in vertebrates. Like the majority of polarity proteins, plasma membrane (PM)/cortical localization of Dlg is required for its function in polarity and tumorigenesis, but the exact mechanisms targeting Dlg to PM remain to be fully elucidated. Here we show that, similar to the recently discovered polybasic polarity proteins such as Lgl and aPKC, Dlg also contains a positively charged polybasic domain that electrostatically binds the PM phosphoinositides PI4P and PI(4,5)P2. Electrostatic targeting by the polybasic domain contributes significantly to the PM localization of Dlg in follicular and early embryonic epithelial cells, and is crucial for Dlg to regulate both polarity and tumorigenesis. The electrostatic PM targeting of Dlg is controlled by a potential phosphorylation-dependent allosteric regulation of its polybasic domain, and is specifically enhanced by the interactions between Dlg and another basolateral polarity protein and tumor suppressor Scrib. Our studies highlight an increasingly significant role of electrostatic PM targeting of polarity proteins in regulating cell polarity.

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Celsr1 and CAMSAP3 differently regulate intercellular and intracellular cilia orientation in oviduct multiciliated cells

10.1101/2020.08.28.273169 ◽

2020 ◽

Author(s):

Fumiko Matsukawa Usami ◽

Masaki Arata ◽

Dongbo Shi ◽

Sanae Oka ◽

Yoko Higuchi ◽

...

Keyword(s):

Cell Polarity ◽

Planar Cell Polarity ◽

Molecular Mechanisms ◽

The Other ◽

Basal Bodies ◽

Generating Functional ◽

Tissue Polarity ◽

Other Hand ◽

Multiciliated Cells ◽

Polarity Regulation

SummaryThe molecular mechanisms by which cilia orientation is coordinated within and between multiciliated cells (MCCs) is not fully understood. By observing the orientation of basal bodies (BB) in MCCs of mouse oviducts, here, we show that Celsr1, a planar cell polarity (PCP) factor involved in tissue polarity regulation, is dispensable for determining BB orientation in individual cells, whereas CAMSAP3, a microtubule minus-end regulator, is critical for this process but not for PCP. MCCs exhibit a characteristic BB orientation and microtubule gradient along the tissue axis, and these intracellular polarities were maintained in the cells lacking Celsr1, although the intercellular coordination of the polarities was partly disrupted. On the other hand, CAMSAP3 regulated the assembly of microtubules interconnecting BBs by localizing at the BBs, and its mutation led to disruption of intracellular coordination of BB orientation, but not affecting PCP factor localization. Thus, both Celsr1 and CAMSAP3 are responsible for BB orientation but in distinct ways; and therefore, their cooperation should be critical for generating functional multiciliated tissues.

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Life and Death of Fungal Transporters Under the Challenge of Polarity

10.20944/preprints202007.0601.v1 ◽

2020 ◽

Author(s):

Sofia Dimou ◽

George Diallinas

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Proteins ◽

Cell Polarity ◽

Secretory Pathway ◽

Fungal Growth ◽

Present Evidence ◽

Life And Death ◽

Early Endosomes ◽

Stress Signals

Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental or stress signals. Sorting of transporters from their site of synthesis, the Endoplasmic Reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the MVB/lysosomes/vacuole system. In specific cases internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review we present evidence that shows that transporter traffic to the PM takes place through Golgi-bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking.

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