Precise segregation of chromosomes requires the activity of a specialized chromatin region, the centromere, that assembles the kinetochore complex to mediate the association with spindle microtubules. We show here that Mal2p, previously identified as a protein required for genome stability, is an essential component of the fission yeast centromere. Loss of functional Mal2p leads to extreme missegregation of chromosomes due to nondisjunction of sister chromatids and results in inviable cells. Mal2p associates specifically with the central region of the complex fission yeast centromere, where it is required for the specialized chromatin architecture as well as for transcriptional silencing of this region. Genetic evidence indicates that mal2 ؉ interacts with mis12 ؉ , encoding another component of the inner centromere core complex. In addition, Mal2p is required for correct metaphase spindle length. Our data imply that the Mal2p protein is required to build up a functional fission yeast centromere.The molecular mechanisms that are required for the faithful segregation of the duplicated sister chromatids in mitosis are necessary for the accurate inheritance of genetic information. Accurate segregation of chromosomes is dependent on the centromere and an associated kinetochore complex. This complex provides the site on the chromosome for the attachment of the mitotic spindle fibres and is also required for a sensing mechanism that signals correct completion of metaphase and thus allows entry into anaphase. In general the centromerekinetochore complex is formed at a single position on the chromosome. The cis-acting DNA present at this site varies in sequence composition and complexity in different eucaryotic organisms. In higher eucaryotes, centromeric DNA consists of highly repetitive regions encompassing up to millions of base pairs (reviewed in reference 13) but the exact sequence requirements for centromere function are not well defined. In contrast, the centromere DNA of the budding yeast Saccharomyces cerevisiae is confined to a very well defined 125-bp region containing highly conserved DNA sequence motifs, which is sufficient to mediate accurate segregation of chromosomes in mitosis and meiosis. In addition, substantial progress has been made in the characterization of budding yeast centromere proteins (reviewed in reference 39). The centromere DNA of the fission yeast Schizosaccharomyces pombe lies in between these two extremes. It is 40 to 120 kb in size and moderately repetitive. The centromere DNA on the three S. pombe chromosomes all consist of a central core region flanked by inverted repeats that can be subdivided into inner and outer repetitive sequences of variable size structure (reviewed in reference 56; see Fig. 4 for cen1). The central core regions have an unusual chromatin architecture that does not show the long-range regular nucleosome ladders (52, 58). This specialized chromatin structure appears to be related to centromere function since mutations that abolish this chromatin structure give r...

Structure±function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter. Transport of solutes across cellular membranes, whether catalyzed by channels or carriers, is necessary for complex metabolism. Although adenine nucleotides, the universal cellular energy currency, fulfil an irreplaceable function, they are viewed as part of the intracellular compartment and often are not transported across biological membranes. However, there are three exceptions to this generalization in which transport of ATP and ADP appropriately occurs: in obligate intracellular bacteria, in mitochondria, and in plastids. In heterotrophic eukaryotic cells, most ATP is synthesized during oxidative phosphorylation and accumulates in the mitochondrial matrix, making it necessary to export this intermediate into the cytosol to fuel anabolic reactions. This transport is mediated by the mitochondrial ADP/ATP carrier (AAC), one of the best-characterized membrane carrier proteins [1±7]. AAC is a member of a large group of transporters comprising the mitochondrial carrier family [3]. These carriers share the same evolutionary ancestor, exhibit an internal sequence triplication of about 100 amino acids, and in the functional state probably form a homodimer from the monomer that has six transmembrane domains [8±10]. A second type of adenylate transporter resides in the inner envelope membrane of plant plastids [11±14]. This transporter displays a high structural similarity to the third type of ATP/ADP transporter, that from rickettsiae [15] and chlamydiae [16], obligate intracellular bacteria that grow within eukaryotic cells. Neither plastidic nor bacterial types show homology to the mitochondrial ADP/ATP carriers and are insensitive to the AAC inhibitors [17]. The plastidic and bacterial ATP/ADP transporters belong to the 12 ...

Structure±function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter.Keywords: ATP/ADP transporter; structure/function relationship; site-specific mutations; plastid; Arabidopsis thaliana.Transport of solutes across cellular membranes, whether catalyzed by channels or carriers, is necessary for complex metabolism. Although adenine nucleotides, the universal cellular energy currency, fulfil an irreplaceable function, they are viewed as part of the intracellular compartment and often are not transported across biological membranes. However, there are three exceptions to this generalization in which transport of ATP and ADP appropriately occurs: in obligate intracellular bacteria, in mitochondria, and in plastids.In heterotrophic eukaryotic cells, most ATP is synthesized during oxidative phosphorylation and accumulates in the mitochondrial matrix, making it necessary to export this intermediate into the cytosol to fuel anabolic reactions. This transport is mediated by the mitochondrial ADP/ATP carrier (AAC), one of the best-characterized membrane carrier proteins [1±7]. AAC is a member of a large group of transporters comprising the mitochondrial carrier family [3]. These carriers share the same evolutionary ancestor, exhibit an internal sequence triplication of about 100 amino acids, and in the functional state probably form a homodimer from the monomer that has six transmembrane domains [8±10].A second type of adenylate transporter resides in the inner envelope membrane of plant plastids [11±14]. This transporter displays a high structural similarity to the third type of ATP/ADP transporter, that from rickettsiae [15] and chlamydiae [16], obligate intracellular bacteria that grow within eukaryotic cells. Neither plastidic nor bacterial types show homology to the mitochondrial ADP/ATP carrier...