THE CDC31P-BINDING PROTEIN KAR1P IS A COMPONENT OF THE HALF-BRIDGE OFTHE YEAST SPINDLE POLE BODY

KAR1 has been identified as an essential gene which is involved in kar yogamy of mating yeast cells and in spindle pole body duplication of m itotic cells (Rose, M. D., and G. R. Fink. 1987. Cell. 48:1047-1060). We investigated the cell cycle-dependent localization of the Kar1 prot ein (Kar1p) and its interaction with other SPB components. Kar1p is as sociated with the spindle pole body during the entire cell cycle of ye ast. 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 b ridge of the spindle pole body. Cdc31p, a Ca2+-binding protein, was pr eviously identified as the first component of the half bridge of the s pindle 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 sequen ce within the carboxy terminal half of Kar1p. The potential Cdc31p-bin ding sequence of Kar1p contains three acidic amino acids which are not found in calmodulin-binding peptides, explaining the different substr ate specificities of Cdc31p and calmodulin. Cdc31p was also able to bi nd to the carboxy terminus of Nuflp/Spc110p, another component of the SPB (Kilmartin, J. V., S. L. Dyes, D. Kershaw, and J. T. Finch. 1993. J. Cell Biol. 123:1175-1184). The association of Kar1p with the spindl e pole body was independent of Cdc31p. Cdc31p, on the other hand, was not associated with SPBs of kar1 cells.