The Cdc31p-Binding Protein Kar1p Is a Component of the Half Bridge of the Yeast Spindle Pole Body

• Published in: The Journal of cell biology Vol. 128; no. 5; pp. 863 - 877
• Main Authors: Spang, Anne, Courtney, Iain, Grein, Katrin, Matzner, Monika, Schiebel, Elmar
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01-03-1995; The Rockefeller University Press
• Subjects: Amino acids; Antibodies; Base Sequence; Calcium-Binding Proteins - metabolism; Cell Compartmentation; Cell cycle; Cell Cycle - physiology; Cell Cycle Proteins; Cells; Cellular biology; Cytoskeletal Proteins; Fluorescent Antibody Technique; Fungal Proteins - genetics; Fungal Proteins - immunology; Fungal Proteins - metabolism; Microscopy; Models, Biological; Molecular Sequence Data; Nuclear membrane; Nuclear Proteins - genetics; Nuclear Proteins - immunology; Nuclear Proteins - metabolism; Plasmids; Polymerase chain reaction; Protein Binding; Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins; Spindle Apparatus - metabolism; Spindle Apparatus - ultrastructure; Spindle pole body; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.128.5.863

KAR1 has been identified as an essential gene which is involved in karyogamy of mating yeast cells and in spindle pole body duplication of mitotic cells (Rose, M. D., and G. R. Fink. 1987. Cell. 48:1047-1060). We investigated the cell cycle-dependent localization of the Kar1 protein (Kar1p) and its interaction with other SPB components. Kar1p is associated with the spindle pole body during the entire cell cycle of yeast. Immunoelectron microscopic studies with anti-Kar1p antibodies or with the monoclonal antibody 12CA5 using an epitope-tagged, functional Kar1p revealed that Kar1p is associated with the half bridge or the bridge of the spindle pole body. Cdc31p, a Ca2+-binding protein, was previously identified as the first component of the half bridge of the spindle pole body (Spang, A., I. Courtney, U. Fackler, M. Matzner, and E. Schiebel. 1993. J. Cell Biol. 123:405-416). Using an in vitro assay we demonstrate that Cdc31p specifically interacts with a short sequence within the carboxy terminal half of Kar1p. The potential Cdc31p-binding sequence of Kar1p contains three acidic amino acids which are not found in calmodulin-binding peptides, explaining the different substrate specificities of Cdc31p and calmodulin. Cdc31p was also able to bind to the carboxy terminus of Nuflp/Spc110p, another component of the SPB (Kilmartin, J. V., S. L. Dyos, D. Kershaw, and J. T. Finch. 1993. J. Cell Biol. 123:1175-1184). The association of Kar1p with the spindle pole body was independent of Cdc31p. Cdc31p, on the other hand, was not associated with SPBs of kar1 cells.