Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore–centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.
When a human cell divides to make new cells, its 46 chromosomes must be replicated and then separated evenly between the two daughter cells. The process of separation is performed by the spindle—a network of fibres that form inside the cell, attach to the chromosomes and pull the copies to the opposite ends of the cell.
The spindle fibres attach to a structure called a kinetochore, which forms at a region of the chromosomes called the centromere. The kinetochore has a layered structure with multiple copies of many proteins, and the inner layer is composed of at least 16 centromeric proteins. These proteins interact directly with the centromere and influence the formation of the rest of the kinetochore and the spindle fibres. While some of the interactions between centromeric proteins have been uncovered, the roles of several of them—including one called CENP-M—remain unknown.
Now, Basilico, Maffini, Weir et al. reveal that CENP-M is essential for assembling and stabilizing the inner layer of the kinetochore. However, while it is structurally and evolutionarily related to enzymes called GTPases, CENP-M is not an enzyme. Instead, the CENP-M protein interacts with three other centromeric proteins to form a complex that becomes part of the inner layer of the kinetochore. Basilico, Maffini, Weir et al. also find that another centromeric protein, CENP-C, appears to start the assembly of the inner layer. This protein then recruits two complexes made of other centromeric proteins to the kinetochore, including the complex that contains CENP-M.
The next challenge will be to reconstitute larger protein complexes that contain more proteins from the inner layer of the kinetochore, so that these assemblies can be studied in greater detail. It will also be important to investigate how CENP-C acts as a scaffold to organize the interface between the kinetochore and the centromere.