scholarly journals Functions and Functional Domains of the GTPase Cdc42p

2000 ◽  
Vol 11 (1) ◽  
pp. 339-354 ◽  
Author(s):  
Keith G. Kozminski ◽  
Ann J. Chen ◽  
Avital A. Rodal ◽  
David G. Drubin
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Structural Model ◽  
Nodal Point ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus ◽  
Ras Superfamily ◽  
Rho Family ◽  
Polarity Regulation

Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of thesecdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (α5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.

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

2003 ◽  
Vol 14 (12) ◽  
pp. 4958-4970 ◽  
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.

scholarly journals Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

2018 ◽  
Vol 115 (46) ◽  
pp. E10859-E10868 ◽  
Author(s):  
Yuwei Li ◽  
Jason A. Junge ◽  
Cosimo Arnesano ◽  
Garrett G. Gross ◽  
Jeffrey H. Miner ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Explant Culture ◽  
Scaffolding Protein ◽  
Fluorescence Lifetime Imaging ◽  
Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

scholarly journals Complexin cooperates with Bruchpilot to tether synaptic vesicles to the active zone cytomatrix

2019 ◽  
Vol 218 (3) ◽  
pp. 1011-1026 ◽  
Author(s):  
Nicole Scholz ◽  
Nadine Ehmann ◽  
Divya Sachidanandan ◽  
Cordelia Imig ◽  
Benjamin H. Cooper ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
Snare Complex ◽  
C Terminus ◽  
Unknown Protein ◽  
Transmission Part ◽  
Molecular Components ◽  
In Vivo Screen

Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.

scholarly journals Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

1999 ◽  
Vol 10 (2) ◽  
pp. 329-344 ◽  
Author(s):  
Martin Dünnwald ◽  
Alexander Varshavsky ◽  
Nils Johnsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Polypeptide Chain ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Living Cells ◽  
C Terminus ◽  
Identical Test ◽  
Split Ubiquitin

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum ofSaccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1

1992 ◽  
Vol 12 (12) ◽  
pp. 5690-5699 ◽  
Author(s):  
Y Matsui ◽  
A Toh-E
Keyword(s):  
Cell Polarity ◽  
Inhibitory Effect ◽  
High Dose ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Formation ◽  
Stabilizing Agents ◽  
Daughter Cells ◽  
Bud Emergence ◽  
Ras Superfamily ◽  
Bud Site

RHO3 and RHO4 are members of the ras superfamily genes of the yeast Saccharomyces cerevisiae and are related functionally to each other. Experiments using a conditionally expressed allele of RHO4 revealed that depletion of both the RHO3 and RHO4 gene products resulted in lysis of cells with a small bud, which could be prevented by the presence of osmotic stabilizing agents in the medium. rho3 rho4 cells incubated in medium containing an osmotic stabilizing agent were rounded and enlarged and displayed delocalized deposition of chitin and delocalization of actin patches, indicating that these cells lost cell polarity. Nine genes whose overexpression could suppress the defect of the RHO3 function were isolated (SRO genes). Two of them were identical with CDC42 and BEM1, bud site assembly genes involved in the process of bud emergence. A high dose of CDC42 complemented the rho3 defect, whereas overexpression of RHO3 had an inhibitory effect on the growth of mutants defective in the CDC24-CDC42 pathway. These results, along with comparison of cell morphology between rho3 rho4 cells and cdc24 (or cdc42) mutant cells kept under the restrictive conditions, strongly suggest that the functions of RHO3 and RHO4 are required after initiation of bud formation to maintain cell polarity during maturation of daughter cells.

scholarly journals Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Roland Chanet ◽  
Dorothée Baïlle ◽  
Marie-Pierre Golinelli-Cohen ◽  
Sylvie Riquier ◽  
Olivier Guittet ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Delta ◽  
Chromosomal Dna ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus

Abstract B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

scholarly journals Coordination of RNA and protein condensation by the P granule protein MEG-3

2020 ◽  
Author(s):  
Helen Schmidt ◽  
Andrea Putnam ◽  
Dominique Rasoloson ◽  
Geraldine Seydoux
Keyword(s):  
Protein Interactions ◽  
Intrinsically Disordered Protein ◽  
Protein Protein Interactions ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
C Terminus ◽  
Necessary And Sufficient

ABSTRACTGerm granules are RNA-protein condensates in germ cells. The mechanisms that drive germ granule assembly are not fully understood. MEG-3 is an intrinsically-disordered protein required for germ (P) granule assembly in C. elegans. MEG-3 forms gel-like condensates on liquid condensates assembled by PGL proteins. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). Using in vitro and in vivo experiments, we find the MEG-3 C-terminus is necessary and sufficient to build MEG-3/PGL co-condensates independent of RNA. The HMGL domain is required for high affinity MEG-3/PGL binding in vitro and for assembly of MEG-3/PGL co-condensates in vivo. The MEG-3 IDR binds RNA in vitro and is required but not sufficient to recruit RNA to P granules. Our findings suggest that P granule assembly depends in part on protein-protein interactions that drive condensation independent of RNA.

scholarly journals Perturbation of BRMS1 interactome reveals pathways that impact metastasis

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259128
Author(s):  
Rosalyn C. Zimmermann ◽  
Mihaela E. Sardiu ◽  
Christa A. Manton ◽  
Md. Sayem Miah ◽  
Charles A. S. Banks ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cancer Metastasis ◽  
Breast Cancer Metastasis ◽  
Metastasis Suppressor ◽  
Protein Protein Interactions ◽  
Multiple Cancer ◽  
C Terminus ◽  
Therapeutic Development

Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes [cell cycle (e.g., CDKN2A), DNA repair (e.g., BRCA1)], and metastasis [(e.g., TCF2 and POLE2)]. Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration in vitro and metastases in vivo. The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1’s molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.

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