scholarly journals Role of a Cdc42p Effector Pathway in Recruitment of the Yeast Septins to the Presumptive Bud Site

2006 ◽  
Vol 17 (3) ◽  
pp. 1110-1125 ◽  
Author(s):  
Masayuki Iwase ◽  
Jianying Luo ◽  
Satish Nagaraj ◽  
Mark Longtine ◽  
Hyong Bai Kim ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Ring Formation ◽  
Cell Cortex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Distinct Structure ◽  
Effector Pathway ◽  
Mutant Phenotypes ◽  
Bud Site

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.

scholarly journals The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

2003 ◽  
Vol 14 (10) ◽  
pp. 4051-4066 ◽  
Author(s):  
Juliane P. Caviston ◽  
Mark Longtine ◽  
John R. Pringle ◽  
Erfei Bi
Keyword(s):  
Ring Formation ◽  
Cell Cortex ◽  
Temperature Sensitive ◽  
Direct Role ◽  
Yeast Saccharomyces Cerevisiae ◽  
Septin Ring ◽  
Gtpase Activating Proteins ◽  
Bud Growth ◽  
Bud Site

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36Gallele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36Gor GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.

Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery

1993 ◽  
Vol 13 (1) ◽  
pp. 399-407
Author(s):  
I J McEwan ◽  
A P Wright ◽  
K Dahlman-Wright ◽  
J Carlstedt-Duke ◽  
J A Gustafsson
Keyword(s):  
Glucocorticoid Receptor ◽  
Protein Interactions ◽  
Core Promoter ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

scholarly journals Functional insight into the role of Orc6 in septin complex filament formation in Drosophila

2015 ◽  
Vol 26 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Katarina Akhmetova ◽  
Maxim Balasov ◽  
Richard P. H. Huijbregts ◽  
Igor Chesnokov
Keyword(s):  
Origin Recognition Complex ◽  
Spatial Organization ◽  
Cell Cortex ◽  
Filament Formation ◽  
Gtp Binding ◽  
Complex Functions ◽  
Septin Filament

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

scholarly journals Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

2021 ◽  
Author(s):  
Supansa Pantoom ◽  
Adam Pomorski ◽  
Katharina Huth ◽  
Christina Hund ◽  
Janine Petters ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Copper Metabolism ◽  
Central Protein ◽  
Potential Binding ◽  
Normal Human ◽  
Autophagy Pathway ◽  
Interaction Regions ◽  
Cellular Copper

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in autophagy pathway, so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

scholarly journals Structural basis of the interplay between α-synuclein and Tau in regulating pathological amyloid aggregation

2020 ◽  
Vol 295 (21) ◽  
pp. 7470-7480 ◽  
Author(s):  
Jinxia Lu ◽  
Shengnan Zhang ◽  
Xiaojuan Ma ◽  
Chunyu Jia ◽  
Zhenying Liu ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Neurodegenerative Diseases ◽  
Direct Interaction ◽  
Structural Basis ◽  
Amyloid Aggregation ◽  
C Terminus ◽  
Amyloid Fibrillation

Amyloid aggregation of pathological proteins is closely associated with a variety of neurodegenerative diseases, and α-synuclein (α-syn) deposition and Tau tangles are considered hallmarks of Parkinson's disease and Alzheimer's disease, respectively. Intriguingly, α-syn and Tau have been found to co-deposit in the brains of individuals with dementia and parkinsonism, suggesting a potential role of cross-talk between these two proteins in neurodegenerative pathologies. Here we show that monomeric α-syn and the two variants of Tau, Tau23 and K19, synergistically promote amyloid fibrillation, leading to their co-aggregation in vitro. NMR spectroscopy experiments revealed that α-syn uses its highly negatively charged C terminus to directly interact with Tau23 and K19. Deletion of the C terminus effectively abolished its binding to Tau23 and K19 as well as its synergistic effect on promoting their fibrillation. Moreover, an S129D substitution of α-syn, mimicking C-terminal phosphorylation of Ser129 in α-syn, which is commonly observed in the brains of Parkinson's disease patients with elevated α-syn phosphorylation levels, significantly enhanced the activity of α-syn in facilitating Tau23 and K19 aggregation. These results reveal the molecular basis underlying the direct interaction between α-syn and Tau. We proposed that this interplay might contribute to pathological aggregation of α-syn and Tau in neurodegenerative diseases.

scholarly journals The role of let-7b in the inhibition of hepatic stellate cell activation by rSjP40

2021 ◽  
Vol 15 (6) ◽  
pp. e0009472
Author(s):  
Xiaolei Sun ◽  
Li Zhang ◽  
Yuting Jiang ◽  
Aihong Li ◽  
Dandan Zhu ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Cell Activation ◽  
Stellate Cell ◽  
Direct Interaction ◽  
Collagen Type I ◽  
Collagen I ◽  
Luciferase Assay ◽  
Type I

Background Hepatic stellate cells (HSCs) are one of the main cell types involved in liver fibrosis induced by many factors, including schistosomes. Previous studies in our lab have shown that recombinant P40 protein from Schistosoma japonicum (rSjP40) can inhibit HSC activation in vitro. Let-7b is a member of the let-7 microRNA family and plays an inhibitory role in a variety of diseases and inflammatory conditions. In this study, we investigated the role of let-7b in the inhibition of HSC activation by rSjP40. Methods Expression of let-7b was detected by quantitative real-time PCR. A dual luciferase assay was used to confirm direct interaction between let-7b and collagen I. We also used western blot to assess protein levels of TGFβRI and collagen type I α1 (COL1A1). Results We found that rSjP40 up-regulates expression of let-7b in HSCs. Let-7b inhibits collagen I expression by directly targeting the 3’UTR region of the collagen I gene. Furthermore, we discovered that let-7b inhibitor partially restores the loss of collagen I expression caused by rSjP40. Conclusion Our research clarifies the role of let-7b in the inhibition of HSC activation by rSjP40 and will provide new insights and ideas for the inhibition of HSC activation and treatment of liver fibrosis.

CD47 Glycoprotein Interacts with p4.1R and p55 in the Erythrocyte.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1126-1126
Author(s):  
Neil D. Avent ◽  
Zoe E. Plummer ◽  
David J. Head
Keyword(s):  
Erythrocyte Membrane ◽  
Direct Interaction ◽  
Potential Interaction ◽  
Erythrocyte Membranes ◽  
Mature Erythrocyte ◽  
Diffuse Fraction ◽  
Cytoplasmic Face ◽  
Free Translation

Abstract CD47 is a 47–50kDa membrane glycoprotein with 5 known isoforms. The role of CD47 within the erythrocyte membrane remains the subject of much research and debate though we recently provided evidence that CD47 may function as an inducer of eryptosis (Head et al, 2005). As both a cytoskeletal linked fraction and a smaller membrane diffuse fraction of CD47 exists, it is most likely that there are a number of protein species that are able to bind to CD47 at its cytoplasmic face. Our research has focused on a study of the molecular interactions of erythrocyte CD47 with erythrocyte membrane skeletal proteins protein 4.1R (p4.1R), protein 4.2 (p4.2) and p55. Here we demonstrate the ubiquitous expression of all CD47 isoforms in haemopoietic cells and tissues using basic and real-time PCR. Via immunoprecipitation of CD47 from mature erythrocyte membranes using the anti-CD47 mAb BRIC-126, yeast two-hybrid analysis and in vitro co-immunoprecipitation of 35[S] labelled peptides in a cell-free translation procedure, a novel ternary complex involving CD47, p55 and p4.1R has been indicated. More specifically, the potential interaction between p55/p4.1R and the cytoplasmic face of CD47 has been localised to the PDZ and FERM domain of these proteins respectively. Though research suggests p4.2 provides the major cytoskeletal attachment of CD47 (Bruce et al, 2002; Mouro-Chanteloup et al, 2003), a direct interaction between CD47 and p4.2 was not suggested by our study and remains undemonstrated. We continue to further the evidence for a functional role of CD47 in eryptosis. We propose p4.1R links CD47 to the apoptotic machinery of the cell and suggest a mechanism whereby cytoskeletal rearrangement and PS exposure occurs. 4.1null cells have been obtained and are currently being investigated. Further characterisation of the eryptotic pathway may offer insight into potential therapies for erythroleukaemia characterised by resistance of the erythroid lineage to apoptosis. This work has equal significance in the stabilization of red cell preparations in the blood transfusion setting.

scholarly journals A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA

2002 ◽  
Vol 184 (14) ◽  
pp. 3992-4002 ◽  
Author(s):  
Akira Maehara ◽  
Seiichi Taguchi ◽  
Tatsuaki Nishiyama ◽  
Tsuneo Yamane ◽  
Yoshiharu Doi
Keyword(s):  
Paracoccus Denitrificans ◽  
Direct Interaction ◽  
Negative Regulator ◽  
Dna Complexes ◽  
Short Chain Length ◽  
Dnase I Footprinting ◽  
Associated Proteins ◽  
Direct Binding

ABSTRACT Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

scholarly journals Interactions among Rax1p, Rax2p, Bud8p, and Bud9p in Marking Cortical Sites for Bipolar Bud-site Selection in Yeast

2004 ◽  
Vol 15 (11) ◽  
pp. 5145-5157 ◽  
Author(s):  
Pil Jung Kang ◽  
Elizabeth Angerman ◽  
Kenichi Nakashima ◽  
John R. Pringle ◽  
Hay-Oak Park
Keyword(s):  
Cell Cortex ◽  
Type I ◽  
Yeast Saccharomyces Cerevisiae ◽  
Daughter Cells ◽  
Division Site ◽  
Distal Pole ◽  
Mother And Daughter ◽  
Mother Cells ◽  
Diploid Cells ◽  
Bud Site

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.

scholarly journals The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1376-1390 ◽  
Author(s):  
G C Chen ◽  
L Zheng ◽  
C S Chan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Selection ◽  
Yeast Cells ◽  
Gtpase Activating Protein ◽  
Lim Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Lim Domains ◽  
Bud Site

Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.

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