Multivalency ensures persistence of a +TIP body at specialized microtubule ends.

Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential for cell division and morphogenesis. Here we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIP proteins Kar9 (functional homologue of APC, MACF and SLAIN), Bim1 (orthologous to EB1) and Bik1 (orthologous to CLIP-170). A multivalent web of redundant interactions links the three +TIPs together to form a '+TIP body' at the end of chosen microtubules.

Structure and regulation of the microtubule plus-end tracking protein Kar9.

In many eukaryotes, coordination of chromosome segregation with cell cleavage relies on the patterned interaction of specific microtubules with actin filaments through dedicated microtubule plus-end tracking proteins (+TIPs). However, how these +TIPs are spatially controlled is unclear. The yeast +TIP Kar9 drives one of the spindle aster microtubules along actin cables to align the mitotic spindle with the axis of cell division.

Structure-Function Relationship of the Bik1-Bim1 Complex.

In budding yeast, the microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex that interacts with partners involved in spindle positioning, including Stu2 and Kar9. Here, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique, functionally relevant CAP-Gly elements, establishing Bik1CG as a specific C-terminal phenylalanine recognition domain.

Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning.

The Kar9 pathway promotes nuclear fusion during mating and spindle alignment during metaphase in budding yeast. How Kar9 supports the different outcome of these two divergent processes is an open question. Here, we show that three sites in the C-terminal disordered domain of Kar9 mediate tight Kar9 interaction with the C-terminal dimerization domain of Bim1 (EB1 orthologue).

Cohesin's concatenation of sister DNAs maintains their intertwining.

The contribution of DNA catenation to sister chromatid cohesion is unclear partly because it has never been observed directly within mitotic chromosomes. Differential sedimentation-velocity and gel electrophoresis reveal that sisters of 26 kb circular minichromosomes are held together by catenation as well as by cohesin. The finding that chemical crosslinking of cohesin's three subunit interfaces entraps sister DNAs of circular but not linear minichromosomes implies that cohesin functions using a topological principle.

Both interaction surfaces within cohesin's hinge domain are essential for its stable chromosomal association.

Background: The cohesin complex that mediates sister chromatid cohesion contains three core subunits: Smc1, Smc3, and Scc1. Heterotypic interactions between Smc1 and Smc3 dimerization domains create stable V-shaped Smc1/Smc3 heterodimers with a hinge at the center and nucleotide-binding domains (NBDs) at the ends of each arm. Interconnection of each NBD through their association with the N- and C-terminal domains of Scc1 creates a tripartite ring, within which sister DNAs are thought to be entrapped (the ring model).

The cohesin ring concatenates sister DNA molecules.

Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings.