| Abstract: | The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation. |
