Genetic interactions between a spindle pole body (SPB) gene kar1, and the PKC1 MAP kinase cascade and nuclear envelope proteins

The most important function of microtubule organizing centers (MTOCs) is to establish the mitotic spindle, the machinery by which chromosomes segregate during cell division. For spindle formation, the MTOC duplicates to establish the two spindle poles. The yeast MTOC is a nuclear envelope embedded structure called the spindle pole body (SPB). My thesis work has identified two sets of interacting genes. One set encodes components of a signal transduction pathway that may be important for regulation of SPB duplication. Another set encodes nuclear envelope proteins that may be important in structural aspects of SPB assembly and its association with the nuclear envelope.; The kar1-Delta17 allele causes a temperature sensitive defect in SPB duplication due to failure to localize yeast centrin/Cdc31p to the SPB. As the core of my thesis work, I performed a synthetic lethal screen with a kar1-Delta17 strain to identify regulatory and structural SPB components that interact with Kar1p and/or Cdc31p. I identified mutations in three different genetic loci, REG1, MPK1, and NEM1, as being synthetically lethal with kar1-Delta17 and further characterized two of the genes, MPK1 and NEM1.; MPK1 is the MAP kinase of the PKC1 cell integrity pathway. Multiple PKC1 pathway components showed genetic interactions specifically with those SPB duplication mutants that interact with CDC31. We provide evidence that the genetic interactions were not due to cell integrity defects of the SPB duplication mutants but rather because PKC1 pathway promotes SPB duplication by acting on CDC31 or a functionally related gene. The study also showed a previously unreported link between SPC110 and CDC31 suggesting that CDC31 and SPC110 have interrelated functions in vivo.; Mutations in NEM1 and SPO7 encoding physically interacting nuclear envelope integral membrane proteins, showed genetic interactions specifically with kar1-Delta17 and not other SPB duplication mutants. Overexpression of NEM1 partially suppressed the temperature sensitive growth defect of kar1-Delta17 , indicating the two proteins had overlapping functions. Genetic analysis suggest that NEM1 is required for residual Kar1-Delta17p function in SPB duplication. To explain the genetic interactions, we propose that Nem1p, and possibly Spo7p, associate with Kar1p and assists in SPB duplication.