scholarly journals A Protein Interaction Map of the Mitotic Spindle

2007 ◽  
Vol 18 (10) ◽  
pp. 3800-3809 ◽  
Author(s):  
Jonathan Wong ◽  
Yuko Nakajima ◽  
Stefan Westermann ◽  
Ching Shang ◽  
Jung-seog Kang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Interactions ◽  
Mitotic Spindle ◽  
Phosphorylation Site ◽  
Protein Functions ◽  
Regulation Mechanisms ◽  
Novel Interactions ◽  
Interaction Map ◽  
Spindle Function ◽  
Two Hybrid

The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities.

scholarly journals The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9p

2006 ◽  
Vol 17 (1) ◽  
pp. 178-191 ◽  
Author(s):  
Jeffrey K. Moore ◽  
Sonia D'Silva ◽  
Rita K. Miller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mitotic Spindle ◽  
Phosphorylation Site ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Basic Domain ◽  
Pole Body ◽  
Two Hybrid ◽  
Insight Into

Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1Δ mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5Δ mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.

scholarly journals Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system

2001 ◽  
Vol 82 (4) ◽  
pp. 935-939 ◽  
Author(s):  
Deyin Guo ◽  
Minna-Liisa Rajamäki ◽  
Mart Saarma ◽  
Jari P. T. Valkonen
Keyword(s):  
Hybrid System ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Yeast Two Hybrid ◽  
Potato Virus A ◽  
Yeast Two Hybrid System ◽  
Multiple Protein ◽  
Interaction Map ◽  
Two Hybrid ◽  
Shed Light

A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus Potyvirus, family Potyviridae) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC–HC, HC–CI, VPg–VPg, NIa–NIb and CP–CP) and weak but reproducible interactions were detected for seven additional combinations (P1–CI, P3–NIb, NIaPro–NIb, VPg–NIa, VPg–NIaPro, NIaPro–NIa and NIa–NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC–HC, VPg–VPg, VPg–NIa, NIa–NIa and NIa–NIb) and weaker but reproducible interactions were detected for three additional combinations (P3–NIa, NIa–NIaPro and CP–CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg–NIa, NIa–NIaPro and NIa–NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.

scholarly journals Novel Roles for Saccharomyces cerevisiae Mitotic Spindle Motors

1999 ◽  
Vol 147 (2) ◽  
pp. 335-350 ◽  
Author(s):  
Frank R. Cottingham ◽  
Larisa Gheber ◽  
Dana L. Miller ◽  
M. Andrew Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Cytoplasmic Dynein ◽  
Spindle Positioning ◽  
Bipolar Spindle ◽  
Accessory Factor ◽  
Spindle Function ◽  
Related Proteins ◽  
Spindle Structure

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.

Protein–protein interactions

2010 ◽  
Vol 38 (4) ◽  
pp. 875-878 ◽  
Author(s):  
Mike P. Williamson ◽  
Michael J. Sutcliffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Present Article ◽  
High Throughput ◽  
Protein Interactions ◽  
Metabolic Pathway ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Good Evidence ◽  
Protein Protein Interactions ◽  
Two Hybrid

In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’ proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

scholarly journals A High-ThroughputCandida albicansTwo-Hybrid System

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Floris Schoeters ◽  
Carol A. Munro ◽  
Christophe d’Enfert ◽  
Patrick Van Dijck
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
High Throughput ◽  
Protein Interactions ◽  
Fungal Pathogen ◽  
Model Organism ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Content Type ◽  
Two Hybrid

ABSTRACTCandida albicansis a human fungal pathogen that does not follow the universal codon usage, as it translates the CUG codon into serine rather than leucine. This makes it difficult to study protein-protein interactions using the standard yeast two-hybrid (Y2H) system in the model organismSaccharomyces cerevisiae. Due to the lack of adapted tools, only a small number of protein-protein interactions (PPIs) have been detected or studied usingC. albicans-optimized tools despite the importance of PPIs to understand cell biology. However, with the sequencing of the whole genome ofC. albicans, the availability of an ORFeome collection containing 5,099 open reading frames (ORFs) in Gateway-adapted donor vectors, and the creation of a Gateway-compatibleC. albicans-specific two-hybrid (C2H) system, it became possible to study protein-protein interactions on a larger scale usingC. albicansitself as the model organism. Erroneous translations are hereby eliminated compared to using theS. cerevisiaeY2H system. Here, we describe the technical adaptations and the first application of the C2H system for a high-throughput screen, thus making it possible to screen thousands of PPIs at once inC. albicansitself. This first, small-scale high-throughput screen, using Pho85 as a bait protein against 1,646 random prey proteins, yielded one interacting partner (Pcl5). The interaction found with the high-throughput setup was further confirmed with a low-throughput C2H experiment and with a coimmunoprecipitation (co-IP) experiment.IMPORTANCECandida albicansis a major fungal pathogen, and due to the rise of fungal infections and emerging resistance to the limited antifungals available, it is important to develop novel and more specific antifungals. Protein-protein interactions (PPIs) can be applied as very specific drug targets. However, because of the aberrant codon usage ofC. albicans, the traditional yeast two-hybrid system inSaccharomyces cerevisiaeis difficult to use, and only a limited number of PPIs have been described inC. albicans. To overcome this, aC. albicanstwo-hybrid (C2H) system was developed in 2010. The current work describes, for the first time, the application of the C2H system in a high-throughput setup. We hereby show the usefulness of the C2H system to investigate and detect PPIs inC. albicans, making it possible to further elucidate protein networks inC. albicans, which has the potential to lead to the development of novel antifungals which specifically disrupt PPIs important for virulence.

scholarly journals Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

2012 ◽  
Vol 11 (11) ◽  
pp. 1167-1176 ◽  
Author(s):  
Se-Hui Jung ◽  
Kangseung Lee ◽  
Deok-Hoon Kong ◽  
Woo Jin Kim ◽  
Young-Myeong Kim ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transglutaminase 2 ◽  
Proteomic Profiling ◽  
Protein Activity ◽  
Protein Arrays ◽  
Protein Functions ◽  
Quantitative Validation ◽  
Interaction Map ◽  
On Chip ◽  
Related Proteins

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.

scholarly journals Split-Ubiquitin Two-Hybrid Assay To Analyze Protein-Protein Interactions at the Endosome: Application to Saccharomyces cerevisiae Bro1 Interacting with ESCRT Complexes, the Doa4 Ubiquitin Hydrolase, and the Rsp5 Ubiquitin Ligase

2007 ◽  
Vol 6 (8) ◽  
pp. 1266-1277 ◽  
Author(s):  
Elina Nikko ◽  
Bruno André
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Single Amino Acid ◽  
Deubiquitinating Enzymes ◽  
Endosomal Membrane ◽  
Rich Domain ◽  
Split Ubiquitin ◽  
Two Hybrid

ABSTRACT Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

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