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© The Rockefeller University Press, 0021-9525/1999//809 $5.00
The Journal of Cell Biology, Volume 145, Number 4, , 1999 809-823

Regular Articles

Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae



Ian R. Adams and John V. Kilmartin

MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.

Key Words: spindle pole body • duplication • Spc42p • Spc29p • Nud1p

Abbreviations used in this paper: bt, bis-tris; GFP, green fluorescent protein; IL, intermediate layer; MALDI, matrix-assisted laser desorption/ ionization; NLS, nuclear localization sequence; SPB, spindle pole body; ts, temperature-sensitive.

We are particularly grateful to Sew Yeu Peak-Chew and Ian Fearnley for the mass spectrometry, Andy Kaja for technical help in strain and plasmid construction, Douglas Kershaw for cutting the serial thin sections, and Anne Donaldson for two of the EM pictures. Thanks are also due to M.S. Robinson for discussion. We apologize to the authors of references which we were unable to include because of length restrictions.


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