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 alpha 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 alpha 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.
Li
Sam
S
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK.
Sandercock
Alan M
AM
Conduit
Paul
P
Robinson
Carol V
CV
Williams
Roger L
RL
Kilmartin
John V
JV
D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D018360:Crystallography, X-Ray; D013058:Mass Spectrometry; D008868:Microtubule Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D020539:Sequence Analysis, Protein; D008941:Spindle Apparatus / Q000502:physiology* / Q000648:ultrastructure
C083769:CDC31 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D008868:Microtubule Proteins; D012097:Repressor Proteins; C121820:SFI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins