Yeast cAMP-dependent protein kinase (PKA) activity is essential for growth and antagonizes induction of the general stress response as well as accumulation of glycogen stores. Previous studies have suggested that the PKA effects on the two latter processes result in part from transcription repression. Here we show that transcription derepression that accompanies PKA depletion is dependent upon the presence of two redundant Zn2+-finger transcription factors, Msn2p and Msn4p. The Msn2p and Msn4p proteins were shown previously to act as positive transcriptional factors in the stress response pathway, and our results suggest that Msn2p and Msn4p also mediate PKA-dependent effects on stress response as well as glycogen accumulation genes. Interestingly, PKA activity is dispensable in a strain lacking Msn2p and Msn4p activity. Thus, Msn2p and Msn4p may antagonize PKAdependent growth by stimulating expression of genes that inhibit growth. In agreement with this model, Msn2p/Msn4p function is required for expression of a gene, YAK1, previously shown to antagonize PKA-dependent growth. These results suggest that Msn2p/Msn4p-dependent gene expression may account for all, or at least most, of the pleiotropic effects of yeast PKA, including growth regulation, response to stress and carbohydrate store accumulation.

Two potential breast cancer susceptibility genes, encoding the BRCA1-interacting proteins ZNF350 (or ZBRK1) and BRIP1 (or BACH1), have been identified in yeast two-hybrid screens. We sequenced these genes in probands from 21 families with potentially inherited breast/ovarian cancer, all of which were negative for BRCA1/BRCA2 mutations. Families had at least one case of male breast cancer, two cases of ovarian cancer, or three or more cases of breast and ovarian cancer. In addition, 58 early-onset (before age 35) breast cancer cases and 30 reference individuals were analyzed. Of 17 variants detected in ZBRK1, a missense mutation Val524Ile was identified in the proband of one high-risk family, but no other family members were available for testing. Of 25 variants identified in BRIP1, in addition to four common silent or missense mutations, we identified Gln540Leu, a non-conservative amino acid change, in a single familial proband with inflammatory breast cancer, but this mutation was not present in her three relatives with breast cancer. Haplotype analysis suggests that all ZBRK1 SNPs fall within a single block with two SNPs capturing 92% of the haplotype diversity, while the BRIP1 SNPs fall in two blocks, with five SNPs capturing 89% of the haplotype diversity. Based on sequencing of ZBRK1 and BRIP1 in 21 BRCA1/2-negative probands from inherited breast/ovarian cancer families, it appears unlikely that mutations in these genes account for a significant fraction of inherited breast cancer. Further analysis in unselected cases will be required to know whether the identified variants play a role in genetic predisposition to breast cancer in the general population. Hum Mutat 22:121-128, 2003. Published 2003 Wiley-Liss, Inc.

Free ␤-tubulin not in heterodimers with ␣-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free ␤-tubulin. This study focused specifically on the function of Rbl2p, which, like ␣-tubulin, can rescue cells from free ␤-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of ␤-tubulin relative to ␣-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of ␤-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to ␤-tubulin. The data suggest that Rbl2p binds transiently to free ␤-tubulin, which then passes into an aggregated form that is not toxic.Studies of cellular control of microtubule assembly have focused primarily on the assembly reaction from ␣/␤-tubulin heterodimers to microtubule polymers and on the identification of protein cofactors and structures that modulate this polymerization (8-10). Results obtained by several approaches suggest that cells may also regulate microtubule morphogenesis at stages preceding the polymerization reaction. Of particular interest are proteins that appear to interact with the ␣-and ␤-tubulin polypeptides and modulate their activities. We are studying these proteins in the yeast Saccharomyces cerevisiae in order to understand their in vivo functions.One of these yeast proteins is Rbl2p. Identified in a search for proteins that, when overexpressed, rescue cells from the toxicity of free ␤-tubulin (5), Rbl2p binds monomeric ␤-tubulin to form a heterodimer that excludes ␣-tubulin, both in vivo and in vitro (5). Pulse-labeling experiments demonstrate that Rbl2p can bind both newly synthesized ␤-tubulin before it is incorporated into ␣/␤-tubulin heterodimers and ␤-tubulin released by dissociation of heterodimers (4). However, the precise function of Rbl2p in vivo is not known.Biochemical experiments with the vertebrate homolog of Rbl2p, cofactor A, suggest one possible function. Cofactor A was purified from extracts based on its activity in an in vitro tubulin-folding assay that monitors the exchange of tubulin polypeptides released from the cytosolic chaperonin Tri-C into preexisting ␣/␤-tubulin heterodimers (14, 30). Five cofactors facilitate this reaction. Three of them-cofactors C, D, and E-are necessary for the reaction. The functions of the other two-cofactors A and B-are a subset of the functions of cofactors D and E, respectively, and are not essential in the assay. However, their presence substantially stimulates the reaction (approximately fourfold for cofactor A [21]).These experiments also suggest a pathway for the exchange reaction between unfolded tubulin polypeptides and heterodimers. When ␤-tubulin polypeptides are released from the cytosolic chaperonin, they are able initially to bind either cofactor A or c...