We have isolated a new yeast gene (PCC1) that codes for a factor homologous to human cancer-testis antigens. We provide evidence that Pcc1p is a new transcription factor and that its mutation affects expression of several genes, some of which are involved in cell cycle progression and polarized growth. Mutation of Pcc1p also affects the expression of GAL genes by impairing the recruitment of the SAGA and Mediator co-activators. We characterize a new complex that contains Pcc1p, a kinase, Bud32p, a putative endopeptidase, Kae1p and two additional proteins encoded by ORFs YJL184w and YMLO36w. Genetic and physical interactions among these proteins strongly suggest that this complex is a functional unit. Chromatin immunoprecipitation experiments and multiple genetic interactions of pcc1 mutants with mutants of the transcription apparatus and chromatin modifying enzymes underscore the direct role of the complex in transcription. Functional complementation experiments indicate that the transcriptional function of this set of genes is conserved throughout evolution. The EMBO Journal (2006) IntroductionTranscription of approximately 10% of the yeast genome occurs in a cell-cycle-dependent manner (Spellman et al, 1998). For almost half of these genes, the peak of transcription takes place around START (Spellman et al, 1998;Horak et al, 2002) when cells enter a new cycle and start budding. Thus, it is not surprising that an efficient transcriptional machinery is required for the passage through START. Indeed, mutation of several factors of the transcriptional apparatus leads to defects in START and the accumulation of unbudded cells (Jahng et al, 1988;Prendergast et al, 1990;Drebot et al, 1993;Walker et al, 1997;Koch et al, 1999). Modulation of gene expression is also critical for yeast conjugation. The response to mating pheromone in Saccharomyces cerevisiae involves the transcriptional activation of roughly 200 genes (Roberts et al, 2000), which ultimately determines cell-cycle arrest in the G1 phase and the formation of mating projections.Transcription starts when specific activators bind to their cognate sites on the DNA and recruit co-activator complexes. Co-activators promote the formation of the preinitiation complex (PIC) containing general transcription factors (GTFs) and RNA polymerase II (RNAPII) at the core promoters. This event determines the initiation of transcription, which is followed by elongation and termination.Transcription through chromatin requires the action of chromatin modifying and chromatin remodeling factors. Covalent modifications of histones include phosphorylation, ubiquitylation, methylation and acetylation (Strahl and Allis, 2000;Jenuwein and Allis, 2001). Acetylation of histone N-terminal tails is generally associated with transcriptional activity and histone acetyltransferases (HAT) are recruited to promoters by transcriptional activators. Although several histone deacetylases (HDAC) contribute to transcriptional repression by interacting with specific DNA-binding repressors, recent ...

Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, is accompanied by multiple mitochondrial alterations. Here, we show that mitochondrial fragmentation and cristae alterations characterize cellular models of HD and participate in their increased susceptibility to apoptosis. In HD cells, the increased basal activity of the phosphatase calcineurin dephosphorylates the pro-fission dynamin related protein 1 (Drp1), increasing its mitochondrial translocation and activation, and ultimately leading to fragmentation of the organelle. The fragmented HD mitochondria are characterized by cristae alterations that are aggravated by apoptotic stimulation. A genetic analysis indicates that correction of mitochondrial elongation is not sufficient to rescue the increased cytochrome c release and cell death observed in HD cells. Conversely, the increased apoptosis can be corrected by manoeuvres that prevent fission and cristae remodelling. In conclusion, the cristae remodelling of the fragmented HD mitochondria contributes to their hypersensitivity to apoptosis.

The EKC/KEOPS yeast complex is involved in telomere maintenance and transcription. The Bud32p and kinase-associated endopeptidase 1 (Kaelp) components of the complex are totally conserved in eukarya and archaea. Their genes are fused in several archaeal genomes, suggesting that they physically interact. We report here the structure of the Methanocaldococcus jannaschii Kae1/Bud32 fusion protein MJ1130. Kae1 is an iron protein with an ASKHA fold and Bud32 is an atypical small RIO-type kinase. The structure MJ1130 suggests that association with Kae1 maintains the Bud32 kinase in an inactive state. We indeed show that yeast Kae1p represses the kinase activity of yeast Bud32p. Extensive conserved interactions between MjKae1 and MjBud32 suggest that Kae1p and Bud32p directly interact in both yeast and archaea. Mutations that disrupt the Kae1p/Bud32p interaction in the context of the yeast complex have dramatic effects in vivo and in vitro, similar to those observed with deletion mutations of the respective components. Direct interaction between Kae1p and Bud32p in yeast is required both for the transcription and the telomere homeostasis function of EKC/KEOPS.

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea-Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca 2؉ pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca 2؉ may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca 2؉ in cell microdomains in which the pump co-segregates with partners that modulate the Ca 2؉ message and transduce it to important cell functions.Decades of study on the mammalian Ca 2ϩ -ATPase of the plasma membrane (the PMCA pump) (1) have revealed many properties that set it apart from the other members of the superfamily of P-type pumps (2). One property that is immediately obvious is the long C-terminal tail, which is the locale of the numerous regulatory processes. Another distinctive property of the pump is the wealth of interactors with which the pump triggers processes of general significance for the cell. The basic information on these and other general aspects of the PMCA pump has been collected in other comprehensive reviews (3-5) and will not be repeated here. In this minireview, we instead look at the pump in a novel perspective, in which the extrusion of Ca 2ϩ will not be considered as a mechanism to regulate bulk cell Ca 2ϩ but will be integrated in a complex array of processes that modulate Ca 2ϩ -dependent processes within the cell (6). We focus on the most important regulatory mechanisms of the pump. We posit that, in many cell types, the quantitative contribution of the pump to the total extrusion of Ca 2ϩ could be minor or even irrelevant. Because the main role of the pump would be to do something else, the conclusion would offer a rationale for the puzzling finding that, in some plasma membranes, the pump coexists with a much more powerful Ca 2ϩ extrusion system, e.g. Na ϩ /Ca 2ϩ exchange in cardiomyocytes (7). Naturally, the function of the pump is still essential to the well being of the cell, as underscored by the causative involvement of its malfunction in disease processes (8).The pump contains 10 transmembrane domains, two main cytosolic loops, and a long cytosolic C-terminal tail (9, 10). Separate genes encode its four basic isoforms: PMCA1 is ubiquitous and has a housekeeping role; PMCA4 is also ubiquitous but is endowed with tissue-specific roles; and PMCA2 and PMCA3 are tissue-restricted, with high levels of expression in neurons. Complex alternative splicing processes at a site in the first cytosolic loop of the pump (site A) and within the C-terminal calmodulin-binding domain (CaM-BD 3 ; site C) generate numerous pum...

The Saccharomyces cerevisiae YGR262c/BUD32 gene, whose disruption causes a severe pleiotropic phenotype, encodes a 261-residue putative protein kinase, piD261, whose structural homologues have been identified in a variety of organisms, including humans, and whose function is unknown. We have demonstrated previously that piD261, expressed in Escherichia coli as a recombinant protein, is a Ser/Thr kinase, as judged by its ability to autophosphorylate and to phosphorylate casein. Here we describe a mutational analysis showing that, despite low sequence similarity, the invariant residues representing the signature of protein kinases are conserved in piD261 and in its structural homologues, but are embedded in an altered context, suggestive of unique mechanistic properties. Especially noteworthy are: (i) three unique inserts of unknown function within the N-terminal lobe, (ii) the lack of a lysyl residue which in all other Ser/Thr kinases participates in the catalytic event by interacting with the transferred ATP gamma-phosphate, and which in piD261 is replaced by a threonine, and (iii) an exceedingly short activation loop including two serines, Ser-187 and Ser-189, whose autophosphorylation accounts for the appearance of an upshifted band upon SDS/PAGE. A mutant in which these serines are replaced by alanines was devoid of the upshifted band and displayed reduced catalytic activity. This would include piD261 in the category of protein kinases activated by phosphorylation, although it lacks the RD (Arg-Asp) motif which is typical of these enzymes.

Yeast piD261/Bud32 belongs to the piD261 family of atypical protein kinases structurally conserved, from Archaea to human. The disruption of its gene is causative of severely defective growth. Its human homologue, PRPK, interacts with and phosphorylates the oncosuppressor p53 protein, which is lacking in yeast. Here we show that on one hand piD261/Bud32 interacts with and phosphorylates human p53 in vitro, on the other hand PRPK can partially complement the phenotype of yeast lacking the gene encoding piD261/Bud32. These data indicate that, despite considerable structural divergence, members of the piD261 family from distantly related organisms display a remarkable functional conservation. ß

Background: Mutations in plasma membrane Ca 2ϩ -ATPase (PMCA) isoform 3 and in laminin subunit 1␣ have previously been linked to ataxic phenotypes. Results: A novel PMCA3 missense mutation co-occurring with a compound heterozygous mutation in laminin subunit 1␣ impaired cellular Ca 2ϩ homeostasis. Conclusion:The two mutations could work synergistically to generate the disease phenotype. Significance: A digenic mechanism could be responsible for this case of cerebellar ataxia.