Analysis of the Saccharomyces Spindle Pole by Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry

Abstract

A highly enriched spindle pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional eleven gene products that had not previously been localized to the spindle pole. Immunoelectron microscopy localized eight of these components to different parts of the spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049–6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

“…Antibodies to both Spc72p and Cnm67p localize to the region of the SPB, where we have detected vertical strands connecting the outer plaque to the IL1 and the IL1 to IL2 (Figure 12; Wigge et al, 1998). It seems likely that the connections observed in our tomograms are, in part, made of these proteins.…”

“…A combination of experimental approaches has improved our understanding of SPB components, and more recently localization studies using immunoelectron microscopy have mapped a number of these proteins to the SPB (Wigge et al, 1998). To date 27 proteins have been identified, and 18 have been localized to subregions of the spindle pole (Rout and Kilmartin, 1990;Spang et al, 1996;Sundberg et al, 1996;Wigge et al, 1998). A high percentage of these proteins have predicted coiled-coil domains, including Spc42p, which forms a central crystalline core (Donaldson and Kilmartin, 1996;Bullitt et al, 1997), and Spc110p, which acts as a molecular spacer between the central and inner plaques (Kilmartin et al, 1993;Kilmartin and Goh, 1996).…”

“…The amino terminus of Spc110p in turn interacts with Spc98p Sundberg and Davis, 1997;Nguyen et al, 1998), which along with Spc97p (Knopp and Schiebel, 1997) and Tub4p (Sobel and Snyder, 1995;Marschall et al, 1996;Spang et al, 1996) make up the ␥-tubulin complex at the MT ends at both the inner and outer plaques. Spc72p and Cnm67p localize to the outer plaque of the SPB (Wigge et al, 1998). Although biochemical and genetic analyses of these SPB components are revealing how components of the SPB interact with one another, a detailed understanding of SPB fine structure and how its components are organized is still in its infancy.…”

“…There it initiates new nuclear MTs and contributes to the process of spindle formation (Byers and Goetsch, 1974;Byers, 1981). A combination of experimental approaches has improved our understanding of SPB components, and more recently localization studies using immunoelectron microscopy have mapped a number of these proteins to the SPB (Wigge et al, 1998). To date 27 proteins have been identified, and 18 have been localized to subregions of the spindle pole (Rout and Kilmartin, 1990;Spang et al, 1996;Sundberg et al, 1996;Wigge et al, 1998).…”

Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

“…A "halfbridge" structure, located on one side of the SPB and within the nuclear membrane, serves as the site of SPB duplication. Genetic and biochemical studies have identified approximately 20 proteins that comprise the SPB complex (1,(6)(7)(8). Using immuno-EM, proteins have been localized to regions of the SPB (core, membrane-associated, half-bridge) or particular layers or plaques of the SPB core (outer, intermediate 1, intermediate 2, central, inner) (6,9) (Figure 1).…”