Molecular dissection of yeast spindle pole bodies by two hybrid, in vitro binding, and co-purification

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pΔC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p243–385), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pΔC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5ΔC-BBP1243–385 under CDC5 promoter control partially complemented the cdc5Δ defect. These data suggest that Bbp1pΔC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

Download Full-text

The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2099 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2099-2106 ◽

Cited By ~ 164

Author(s):

R Jiang ◽

M Carlson

Keyword(s):

Protein Kinase ◽

Regulatory Domain ◽

Glucose Starvation ◽

Binding Studies ◽

Triple Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

In Vitro Binding ◽

Vitro Binding ◽

Two Hybrid

The Snf1 protein kinase plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae. Previously, we showed that two-hybrid interaction between Snf1 and its activating subunit, Snf4, is inhibited by high levels of glucose. These findings, together with biochemical evidence that Snf1 and Snf4 remain associated in cells grown in glucose, suggested that another protein (or proteins) anchors Snf1 and Snf4 into a complex. Here, we examine the possibility that a family of proteins, comprising Sip1, Sip2, and Gal83, serves this purpose. We first show that the fraction of cellular Snf4 protein that is complexed with Snf1 is reduced in a sip1delta sip2delta gal83delta triple mutant. We then present evidence that Sip1, Sip2, and Gal83 each interact independently with both Snf1 and Snf4 via distinct domains. A conserved internal region binds to the Snf1 regulatory domain, and the conserved C-terminal ASC domain binds to Snf4. Interactions were mapped by using the two-hybrid system and were confirmed by in vitro binding studies. These findings indicate that the Sip1/Sip2/Gal83 family anchors Snf1 and Snf4 into a complex. Finally, the interaction of the yeast Sip2 protein with a plant Snf1 homolog suggests that this function is conserved in plants.

Download Full-text

The Regulation of Microtubule Dynamics in Saccharomyces cerevisiae by Three Interacting Plus-End Tracking Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e05-09-0892 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2789-2798 ◽

Cited By ~ 59

Author(s):

Michael J. Wolyniak ◽

Kristina Blake-Hodek ◽

Karena Kosco ◽

Eric Hwang ◽

Liru You ◽

...

Keyword(s):

Microtubule Dynamics ◽

Cellular Structures ◽

Binding Assays ◽

Spindle Microtubule ◽

Functional Interactions ◽

In Vitro Binding ◽

Vitro Binding ◽

Cytoplasmic Microtubules ◽

Two Hybrid

Microtubule plus-end tracking proteins (+TIPs) are a diverse group of molecules that regulate microtubule dynamics and interactions of microtubules with other cellular structures. Many +TIPs have affinity for each other but the functional significance of these associations is unclear. Here we investigate the physical and functional interactions among three +TIPs in S. cerevisiae, Stu2, Bik1, and Bim1. Two-hybrid, coimmunoprecipitation, and in vitro binding assays demonstrate that they associate in all pairwise combinations, although the interaction between Stu2 and Bim1 may be indirect. Three-hybrid assays indicate that these proteins compete for binding to each other. Thus, Stu2, Bik1, and Bim1 interact physically but do not appear to be arranged in a single unique complex. We examined the functional interactions among pairs of proteins by comparing cytoplasmic and spindle microtubule dynamics in cells lacking either one or both proteins. On cytoplasmic microtubules, Stu2 and Bim1 act cooperatively to regulate dynamics in G1 but not in preanaphase, whereas Bik1 acts independently from Stu2 and Bim1. On kinetochore microtubules, Bik1 and Bim1 are redundant for regulating dynamics, whereas Stu2 acts independently from Bik1 and Bim1. These results indicate that interactions among +TIPS can play important roles in the regulation of microtubule dynamics.

Download Full-text

Targeting of the Yeast Ty5 Retrotransposon to Silent Chromatin Is Mediated by Interactions between Integrase and Sir4p

Molecular and Cellular Biology ◽

10.1128/mcb.21.19.6606-6614.2001 ◽

2001 ◽

Vol 21 (19) ◽

pp. 6606-6614 ◽

Cited By ~ 98

Author(s):

Weiwu Xie ◽

Xiaowu Gai ◽

Yunxia Zhu ◽

David C. Zappulla ◽

Rolf Sternglanz ◽

...

Keyword(s):

Silent Chromatin ◽

Amino Acid Residues ◽

Binding Assays ◽

Yeast Two Hybrid ◽

In Vitro Binding ◽

C Terminus ◽

Vitro Binding ◽

Primary Determinant ◽

Two Hybrid

ABSTRACT The Ty5 retrotransposons of Saccharomyces cerevisiaeintegrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR andHML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.

Download Full-text

PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis

Molecular Biology of the Cell ◽

10.1091/mbc.e16-09-0686 ◽

2017 ◽

Vol 28 (3) ◽

pp. 488-499 ◽

Cited By ~ 48

Author(s):

Emil D. Parvanov ◽

Hui Tian ◽

Timothy Billings ◽

Ruth L. Saxl ◽

Catrina Spruce ◽

...

Keyword(s):

Yeast Two Hybrid ◽

In Vitro Binding ◽

Recombination Hotspots ◽

Krab Domain ◽

Vitro Binding ◽

Spatial Environment ◽

Genomic Regions ◽

Two Hybrid ◽

Limited Period

In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and coimmunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9’s KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are coexpressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.

Download Full-text

Discovery of New AKT1 Inhibitors by Combination of In silico Structure Based Virtual Screening Approaches and Biological Evaluations

10.26434/chemrxiv.7591202 ◽

2019 ◽

Author(s):

Filip Fratev ◽

Denisse A. Gutierrez ◽

Renato J. Aguilera ◽

suman sirimulla

Keyword(s):

Virtual Screening ◽

Success Rate ◽

Protein Interactions ◽

In Silico ◽

Biological Data ◽

In Vitro Binding ◽

Vitro Binding ◽

Screening Protocol ◽

Screening Approaches

AKT1 is emerging as a useful target for treating cancer. Herein, we discovered a new set of ligands that inhibit the AKT1, as shown by in vitro binding and cell line studies, using a newly designed virtual screening protocol that combines structure-based pharmacophore and docking screens. Taking together with the biological data, the combination of structure based pharamcophore and docking methods demonstrated reasonable success rate in identifying new inhibitors (60-70%) proving the success of aforementioned approach. A detail analysis of the ligand-protein interactions was performed explaining observed activities.<br>

Download Full-text

Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0278 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4317-4326 ◽

Cited By ~ 39

Author(s):

Hiroshi Qadota ◽

Kristina B. Mercer ◽

Rachel K. Miller ◽

Kozo Kaibuchi ◽

Guy M. Benian

Keyword(s):

Caenorhabditis Elegans ◽

Binding Assays ◽

Lim Domain ◽

Yeast Two Hybrid ◽

Lim Domains ◽

Thick Filaments ◽

Vitro Binding ◽

Two Hybrid ◽

Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

Download Full-text

In Vitro Binding of Manganese to Serum Transferrin in Cattle

Acta Veterinaria Scandinavica ◽

10.1186/bf03547829 ◽

1967 ◽

Vol 8 (3) ◽

pp. 228-233

Author(s):

B. Panić

Keyword(s):

Serum Transferrin ◽

In Vitro Binding ◽

Vitro Binding

Download Full-text

ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

Biochemical Journal ◽

10.1042/bj20040590 ◽

2004 ◽

Vol 385 (1) ◽

pp. 309-317 ◽

Cited By ~ 23

Author(s):

Zhefeng ZHAO ◽

Joanna GRUSZCZYNSKA-BIEGALA ◽

Anna ZOLKIEWSKA

Keyword(s):

C2c12 Myotubes ◽

Post Translational Modification ◽

Integrin Β1 ◽

Adp Ribosylation ◽

In Vitro Binding ◽

Vitro Binding ◽

C2c12 Skeletal Muscle Cells ◽

Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

Download Full-text