Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved

Role of the α-Amino Groups of the α and β Chains of Human Hemoglobin in Oxygen-linked Binding of Carbon Dioxide

Abstract Human hemoglobin has been reacted with potassium cyanate and purified to yield three species, α2cβ2c,α2cβ2, and α2β2c, where superscript c denotes specific reaction of cyanate with the α-amino group of the particular chain. The effect of carbon dioxide on the oxygen affinity of these species in the presence and in the absence of 2,3-diphosphoglycerate has been measured. Carbon dioxide has no effect on the oxygen affinity of α2cβ2c, confirming that the usual lowering of the oxygen affinity of carbon dioxide in normal hemoglobin is mediated by the α-amino groups of the α and β chains. The lowering of the oxygen affinity of α2β2c by carbon dioxide was not affected by the presence or absence of 2,3-diphosphoglycerate, showing that 2,3-diphosphoglycerate does not interfere with the oxygen-linked binding of carbon dioxide at the α chain α-amino group. In α2cβ2 there was a much larger effect of carbon dioxide on the oxygen affinity in the absence of 2,3-diphosphoglycerate than in α2β2c; however, on addition of 2,3-diphosphoglycerate the effect of carbon dioxide on the oxygen affinity of α2cβ2 was much smaller and similar to that occurring in α2β2c. This shows that there is a large difference in the carbon dioxide binding constants of the β chain α-amino group in the oxy and deoxy forms of human hemoglobin, and that 2,3-diphosphoglycerate suppresses this difference, probably by binding strongly to the β chain α-amino group of deoxyhemoglobin and displacing any bound carbon dioxide

Structural role of Sfi1p–centrin filaments in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–centrin complexes containing several repeats show Sfi1p as an α helix with centrins wrapped around each repeat and similar centrin–centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly

hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends

CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response

Two domains of centrosomal protein CDK5RAP2, CNN1 and CNN2, link centrosomes to mitotic spindle poles. CNN1 lacking centrosomes are unable to recruit pericentriolar matrix components that mediate attachment to spindle poles