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Full-Text Articles in Life Sciences
A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Oliver Kerscher, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban
A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Oliver Kerscher, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban
Oliver Kerscher
The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a …
Budding Yeast Protein Extraction And Purification For The Study Of Function, Interactions, And Post-Translational Modifications, Eva P. Szymanski, Oliver Kerscher
Budding Yeast Protein Extraction And Purification For The Study Of Function, Interactions, And Post-Translational Modifications, Eva P. Szymanski, Oliver Kerscher
Oliver Kerscher
Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. …
Tim18p Is A New Component Of The Tim54p-Tim22p Translocon In The Mitochondrial Inner Membrane, Oliver Kerscher, Naresh B. Sepuri, Robert E. Jensen
Tim18p Is A New Component Of The Tim54p-Tim22p Translocon In The Mitochondrial Inner Membrane, Oliver Kerscher, Naresh B. Sepuri, Robert E. Jensen
Oliver Kerscher
The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitivetim54-1 mutant. We identified a new gene,TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress thetim54-1 growth defect; thetim18::HIS3 disruption is synthetically lethal …
Recognizing Chromosomes In Trouble: Association Of The Spindle Checkpoint Protein Bub3p With Altered Kinetochores And A Unique Defective Centromere, Oliver Kerscher, Luciana B. Crotti, Munira A. Basrai
Recognizing Chromosomes In Trouble: Association Of The Spindle Checkpoint Protein Bub3p With Altered Kinetochores And A Unique Defective Centromere, Oliver Kerscher, Luciana B. Crotti, Munira A. Basrai
Oliver Kerscher
Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G2/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested …
Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Flup, Josh La, Wei-Chun Au, Nagesh Pasupala, Ruben Levy-Meyers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai
Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Flup, Josh La, Wei-Chun Au, Nagesh Pasupala, Ruben Levy-Meyers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai
Oliver Kerscher
Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and …
The Yeast Hex3·Slx8 Heterodimer Is A Ubiquitin Ligase Stimulated By Substrate Sumoylation, Yang Xie, Oliver Kerscher, Mary B. Kroetz, Heather F. Mcconchie, Patrick Sung, Mark Hochstrasser
The Yeast Hex3·Slx8 Heterodimer Is A Ubiquitin Ligase Stimulated By Substrate Sumoylation, Yang Xie, Oliver Kerscher, Mary B. Kroetz, Heather F. Mcconchie, Patrick Sung, Mark Hochstrasser
Oliver Kerscher
Hex3 and Slx8 are Saccharomyces cerevisiae proteins with important functions in DNA damage control and maintenance of genomic stability. Both proteins have RING domains at their C termini. Such domains are common in ubiquitin and ubiquitin-like protein ligases (E3s), but little was known about the molecular functions of either protein. In this study we identified HEX3 as a high-copy suppressor of a temperature-sensitive small ubiquitin-related modifier (SUMO) protease mutant, ulp1ts, suggesting that it may affect cellular SUMO dynamics. Remarkably, even a complete deletion of ULP1 is strongly suppressed. Hex3 forms a heterodimer with Slx8. We found that the Hex3·Slx8 …
The Yeast Nuclear Pore Complex Functionally Interacts With Components Of The Spindle Assembly Checkpoint, Tatiana Louk, Oliver Kerscher, Robert J. Scott, Munira A. Basrai, Richard W. Wozniak
The Yeast Nuclear Pore Complex Functionally Interacts With Components Of The Spindle Assembly Checkpoint, Tatiana Louk, Oliver Kerscher, Robert J. Scott, Munira A. Basrai, Richard W. Wozniak
Oliver Kerscher
Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p–Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p …