Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin.

e Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the diseaserelevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.

Enological strains of Saccharomyces cerevisiae display a high level of chromosome length polymorphism, but the molecular basis of this phenomenon has not yet been clearly defined. In order to gain further insight into the molecular mechanisms responsible for the karyotypic variability, we examined the chromosomal constitution of a strain known to possess aberrant chromosomes. Our data revealed that the strain carries four rearranged chromosomes resulting from two reciprocal translocations between chromosomes III and I, and chromosomes III and VII. The sizes of the chromosomal fragments exchanged through translocation range from 40 to 150 kb. Characterization of the breakpoints indicated that the translocations involved the RAHS of chromosome III, a transposition hot-spot on the right arm of chromosome I and a region on the left arm of chromosome VII. An analysis of the junctions showed that in all cases Ty elements were present and suggested that the translocations result from recombination between transposable Ty elements. The evidence for multiple translocations mediated by Ty elements in a single strain suggests that spontaneous Ty-driven rearrangement could be quite common and may play a major role in the alteration of karyotypes in natural and industrial yeasts.

Saccharomyces cerevisiae PAU genes constitute the largest multigene family in yeast, with 23 members located mainly in subtelomeric regions. The role and regulation of these genes were previously unknown. We detected PAU gene expression during alcoholic fermentation. An analysis of PAU gene regulation using PAU–lacZ fusions and Northern analyses revealed that they were regulated by anaerobiosis. PAU genes display, however, different abilities to be induced by anaerobiosis and this appears to be related to their chromosomal localization; two subtelomeric copies are more weakly inducible than an interstitial one. We show that PAU genes are negatively regulated by oxygen and repressed by haem. Examination of PAU gene expression in rox1Δ and tup1Δ strains indicates that PAU repression by oxygen is mediated by an unknown, haem‐dependent pathway, which does not involve the Rox1p anaerobic repressor but requires Tup1p. Given the size of the gene family, PAU genes could be expected to be important during yeast life and some of them probably help the yeast to cope with anaerobiosis.

Members of the highly conserved pleiotropic CK1 family of serine/threonine-specific kinases are tightly regulated in the cell and play crucial regulatory roles in multiple cellular processes from protozoa to human. Since their dysregulation as well as mutations within their coding regions contribute to the development of various different pathologies, including cancer and neurodegenerative diseases, they have become interesting new drug targets within the last decade. However, to develop optimized CK1 isoform-specific therapeutics in personalized therapy concepts, a detailed knowledge of the regulation and functions of the different CK1 isoforms, their various splice variants and orthologs is mandatory. In this review we will focus on the stressinduced CK1 isoform delta (CK1δ), thereby addressing its regulation, physiological functions, the consequences of its deregulation for the development and progression of diseases, and its potential as therapeutic drug target.

Membrane potential generation via malate/lactate exchange catalyzed by the malate carrier (MleP) of Lactococcus lactis, together with the generation of a pH gradient via decarboxylation of malate to lactate in the cytoplasm, is a typical example of a secondary proton motive force-generating system. The mleP gene was cloned, sequenced, and expressed in a malolactic fermentation-deficient L. lactis strain. Functional analysis revealed the same properties as observed in membrane vesicles of a malolactic fermentation-positive strain. MleP belongs to a family of secondary transporters in which the citrate carriers from Leuconostoc mesenteroides (CitP) and Klebsiella pneumoniae (CitS) are found also. CitP, but not CitS, is also involved in membrane potential generation via electrogenic citrate/lactate exchange. MleP, CitP, and CitS were analyzed for their substrate specificity. The 2-hydroxycarboxylate motif R 1 R 2 COHCOOH, common to the physiological substrates, was found to be essential for transport although some 2-oxocarboxylates could be transported to a lesser extent. Clear differences in substrate specificity among the transporters were observed because of different tolerances toward the R substituents at the C2 atom. Both MleP and CitP transport a broad range of 2-hydroxycarboxylates with R substituents ranging in size from two hydrogen atoms (glycolate) to acetyl and methyl groups (citromalate) for MleP and two acetyl groups (citrate) for CitP. CitS was much less tolerant and transported only citrate and at a low rate citromalate. The substrate specificities are discussed in the context of the physiological function of the transporters.

The spindle checkpoint delays anaphase onset until all chromosomes are correctly attached to microtubules. Ipl1 protein kinase (Aurora B) is required to correct inappropriate kinetochore-microtubule attachments and for the response to lack of tension between sister kinetochores. Here we identify residues in the checkpoint protein Mad3p that are phosphorylated by Ipl1p. When phosphorylation of Mad3p at two sites is prevented, the cell's response to reduced kinetochore tension is dramatically curtailed. Our data provide strong evidence for a distinct checkpoint pathway responding to lack of sister kinetochore tension, in which Ipl1p-dependent phosphorylation of Mad3p is a key step.Supplemental material is available at http://www.genesdev.org.Received March 2, 2007; revised version accepted March 19, 2007. Since errors in chromosome segregation lead to aneuploidy, cell death, and disease, cells have evolved mechanisms to ensure that their replicated chromosomes are accurately segregated. The spindle checkpoint, involving a conserved network of Mad and Bub proteins, acts as a surveillance system to monitor kinetochore-microtubule interactions during chromosome alignment on the mitotic spindle (Musacchio and Hardwick 2002;Lew and Burke 2003). When activated, it acts to inhibit Cdc20p, an accessory factor for the mitotic E3 ubiquitin ligase known as the Anaphase-Promoting Complex or Cyclosome (APC/C) (Zachariae and Nasmyth 1999; Yu 2002), preventing ubiquitylation of securin and mitotic cyclins and thereby delaying anaphase onset. Once biorientation is achieved (i.e., sister chromatids are attached to opposite poles of the spindle), the checkpoint is satisfied and anaphase can proceed.Biorientation defects can be due to lack of kinetochore-microtubule attachment or result from attachment of both sister kinetochores to the same spindle pole (syntelic or monopolar attachment). Syntelic attachments are thought to result in a lack of tension between sister kinetochores, and error correction mechanisms are required to promote biorientation. It is clear from both vertebrate and yeast studies that Aurora B kinase (Ipl1p in yeast) has a crucial role to play in promoting such biorientation, and that this involves breaking incorrect (syntelic) microtubule attachments (Tanaka et al. 2002;Hauf et al. 2003;Dewar et al. 2004;Lampson et al. 2004). Aurora kinase may also link the correction of inappropriate attachments with a spindle checkpoint-dependent mitotic delay, as it is required to delay anaphase in response to the lack of cohesion at sister centromeres, reduced microtubule dynamics (taxol), and certain kinetochore defects (Biggins and Murray 2001; Ditchfield et al. 2003;Hauf et al. 2003;Pinsky et al. 2003). However, the existence of a distinct "tension checkpoint" mechanism remains controversial (for a recent review, see Pinsky and Biggins 2005). In budding yeast, although in some circumstances Ipl1p can activate the spindle checkpoint through generation of unattached kinetochores when it promotes breakage of defective m...