How to activate p53.

The tumour suppressor protein p53 is stabilised and activated in response to ionising radiation. This is known to depend on the kinase ATM; recent results suggest ATM acts via the downstream kinase Chk2/hCds1, which stabilises p53 at least in part by direct phosphorylation of residue serine 20.

Characterization of Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1 and Rad9.

Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9(+) locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.

The COP9/signalosome complex is conserved in fission yeast and has a role in S phase.

The COP9/signalosome complex is conserved from plant to mammalian cells. In Arabidopsis, it regulates the nuclear abundance of COP1, a transcriptional repressor of photomorphogenic development [1] [2]. All COP (constitutive photomorphogenesis) mutants inappropriately express genes that are normally repressed in the dark. Eight subunits (Sgn1-Sgn8) of the homologous mammalian complex have been purified [3] [4]. Several of these have been previously identified through genetic or protein interaction screens. No coherent model for COP9/signalosome function has yet emerged, but a relationship with cell-cycle progression by transcriptional regulation, protein localisation or protein stability is possible. Interestingly, the COP9/signalosome subunits possess domain homology to subunits of the proteasome regulatory lid complex [5] [6]. Database searches indicate that only Sgn5/JAB1 is present in Saccharomyces cerevisiae, precluding genetic analysis of the complex in cell-cycle regulation. Here we identify a subunit of the signalosome in the fission yeast Schizosaccharomyces pombe through an analysis of the DNA-integrity checkpoint. We provide evidence for the conservation of the COP9/signalosome complex in fission yeast and demonstrate that it functions during S-phase progression.

DNA structure checkpoint pathways in Schizosaccharomyces pombe.

The response to DNA damage includes a delay to progression through the cell cycle to aid DNA repair. Incorrectly replicated chromosomes (replication checkpoint) or DNA damage (DNA damage checkpoint) delay the onset of mitosis. These checkpoint pathways detect DNA perturbations and generate a signal. The signal is amplified and transmitted to the cell cycle machinery. Since the checkpoint pathways are essential for genome stability, the related proteins which are found in all eukaryotes (from yeast to mammals) are expected to have similar functions to the yeast progenitors. This review article focuses on the function of checkpoint proteins in the model system Schizosaccharomyces pombe. Checkpoint controls in Saccharomyces cerevisiae and mammalian cells are mentioned briefly to underscore common or diverse features.

Alteration of substrate affinities and specificities of the Chlorella Hexose/H+ symporters by mutations and construction of chimeras.

The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increased Km value for D-glucose were isolated. The 40-fold increase in Km of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in Km for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km for D-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.

Post-translational fate of CAN1 permease of Saccharomyces cerevisiae.

To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. it was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones. Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition.

Onset of gluconate-H+ symport in Schizosaccharomyces pombe is regulated by the kinases Wis1 and Pka1, and requires the gti1+ gene product.

In the fission yeast Schizosaccharomyces pombe, glucose represses onset of gluconate-H+ symport and inhibits transiently the activity of the symport protein. Wild-type cells harvested from high glucose medium take up gluconate very slowly and the rate of uptake is increased 150-fold in response to glucose starvation. Here it is shown that an intact cAMP cascade is necessary to prevent premature onset in the presence of high glucose concentrations. Cells which have lost either adenylate cyclase (Cyr1) or cAMP-dependent protein kinase (Pka1) transport gluconate up to 60-fold faster than wild-type cells when harvested from high glucose medium. Moreover, inactivation of the stress-sensing Wis1-Sty1 MAP kinase pathway, by loss of Wis1 MAP kinase kinase, diminishes 10-fold the onset of gluconate uptake in response to starvation. A mutant was identified showing a comparable phenotype. By complementation, the gti1+ (gluconate transport inducer 1) gene has been isolated. Disruption of gti1 reduces starvation-induced onset by a similar factor to that observed in wis1 delta cells. Cells over-expressing gti1+ induce gluconate uptake much faster resulting in a threefold higher uptake rate, although gti1+ does not code for the gluconate transport protein. In contrast to the repression of onset, transient downregulation of the gluconate symporter is independent of Pka1 activity and requires ongoing glucose influx. Addition of glucose to starved cyr1 delta cells reduces uptake 9-fold, whereas starved pka1 delta cells, which are able to synthesise cAMP, respond with a 60-fold decrease in transport.

Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro.

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose-proton symporter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of D-glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-beta-D-glucoside in the presence of Escherichia coli L-alpha-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H(+)-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate D-glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of D-glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.

The activity of the gluconate-H+ symporter of Schizosaccharomyces pombe cells is down-regulated by D-glucose and exogenous cAMP.

Schizosaccharomyces pombe cells take up D-gluconate, as an alternative carbon source for growth, during glucose starvation or when cultured on glycerol-containing medium. Gluconate uptake is not detectable while cells are growing logarithmically on glucose. The addition of D-glucose as well as its non-metabolizable analogues to glycerol-grown cells causes an immediate loss of gluconate transport within 1 min. The reversible down-regulation of the gluconate carrier occurs after glucose has been internalized. This regulation is triggered not only by D-glucose but also by extracellular cAMP even in the absence of the cAMP-dependent protein kinase (PKA1).

MEMBRANE TRANSPORT CARRIERS.

Plant and fungal membrane proteins catalyzing the transmembrane translocation of small molecules without directly using ATP or acting as channels are discussed in this review. Facilitators, ion-cotransporters, and exchange translocators mainly for sugars, amino acids, and ions that have been cloned and characterized from Saccharomyces cerevisiae and from various plant sources have been tabulated. The membrane topology and structure of the most extensively studied carriers (lac permease of Escherichia coli, Glut1 of man, HUP1 of Chlorella) are discussed in detail as well as the kinetic analysis of specific Na+ and H+ cotransporters. Finally, the knowledge concerning regulatory phenomena of carriers-mainly of S. cerevisiae-is summarized.

Hexose/H+ symporters in lower and higher plants.

A well-studied transporter of plant cells is the hexose/H+ symporter of the unicellular alga Chlorella kessleri. Its properties, studied in vivo, are briefly summarized. In part, they are atypical and it has been suggested that this porter acts in an asymmetric way. Three genes coding for Chlorella hexose transport activity have been identified (HUP1, HUP2 and HUP3). HUP1 cDNA expressed in a mutant of Schizosaccharomyces pombe not transporting any D-glucose has been studied in detail. Several mutants with changed Km values for substrate were obtained, some by random polymerase chain reaction mutation and selection for decreased sensitivity towards the toxic sugar 2-deoxyglucose, some by site-directed mutagenesis. The amino acids affected clustered in the centre of the putative transmembrane helices V, VII and XI. Large families of hexose transporter genes are found in higher plants (Arabidopsis, Chenopodium, Ricinus). Their functional role is discussed. Finally, the progress made in studying plant transporters in a vesicle system energized by cytochrome c oxidase is summarized.

Km mutants of the Chlorella monosaccharide/H+ cotransporter randomly generated by PCR.

The HUP1 gene codes for the monosaccharide/H+ cotransporter protein of Chlorella kessleri. The gene is functionally expressed in Schizosaccharomyces pombe. This heterologous system has been used to screen for Km mutants of the Chlorella symporter. Since S. pombe transformed with HUP1 cDNA showed a 1000-fold increase in sensitivity toward the toxic sugar analogue 2-deoxyglucose, we screened for transformants with a decreased 2-deoxyglucose sensitivity. The transformants were produced with HUP1 cDNA randomly mutagenized by PCR. From 73 transformants with decreased 2-deoxyglucose sensitivity, four mutants with increased Km values for D-glucose were obtained. The amino acid exchanges responsible for the increased Km values are located in the center of the putative transmembrane helices V (Q179E), VII (Q298R), and XI (V433L/N436Y). Q179N and Q299N had previously been shown by directed mutagenesis to affect the Km value of the transporter for D-glucose. The drastic mutational changes Q298R and N436Y gave rise to very high Km values; however, the corresponding conservative amino acid changes Q298N or N436Q obtained by directed mutagenesis also result in Km values increased by a factor of 10 or 20, respectively. The data therefore support the proposal that at least helices V, VII, and XI may line the sugar translocation path and determine its specificity. These results are discussed in relation to other sugar transporters and to the interaction of the yeast hexokinase B with D-glucose as known from published crystal structures.

The HUP1 gene product of Chlorella kessleri: H+/glucose symport studied in vitro.

An in vitro system was established to measure secondary active transport mediated by plant H+ symporters. For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes. After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative). Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene. The Km value for D-glucose uptake was 5 x 10(-5) M. The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters. In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation. The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.

Structure/function relationship of the Chlorella glucose/H+ symporter.

The Clorella kessleri HUP 1 gene coding for a hexose/H+ symporter has been expressed in a glucose uptake-deficient mutant of Schizosaccharomyces pombe. The transformants are able to grow on glucose and to accumulate 3-O-methylglucose 100-fold. This system has been used to test the activity of specifically mutated HUP 1 cDNAs. All three histidyl residues were exchanged with arginine (H73R, H170R, and H495R) without a major effect on transport activity. When Asp-44 within the first transmembrane helix was replaced by Asn, the transporter was inactive; replacement by Glu (D44E) resulted in a loss of activity by 90% and a 15-fold increased Km value. Glutamine residues conserved in all glucose transporters sequenced so far were exchanged: Q179N (in helix 5), Q298G and Q299N (both in helix 7). Whereas Q298G only resulted in a small Km change, both Q179N and Q299N showed an increase in Km by a factor of 10. Inserting 4 additional amino acids each into the two largest loops (1 and 6) reduced the activity dramatically; only in the latter case this was due to decreased protein synthesis or stability. Two COOH-terminal deletions (-27 and -43 amino acids) were also tested. The 27 COOH-terminal amino acids, but not the 43 COOH-terminal amino acids, could be removed without affecting transporter activity.

Unidirectional arginine transport in reconstituted plasma-membrane vesicles from yeast overexpressing CAN1.

Amino acids are accumulated in Saccharomyces cerevisiae by strictly unidirectional influx systems. To see whether cellular compartmentation causes this unusual amino-acid-transport behaviour, arginine transport was studied in plasma-membrane vesicles. The arginine permease gene CAN1 was overexpressed in S. cerevisiae RH218a and in a permease-deficient mutant RS453 (can1). Reconstituted plasma-membrane vesicles from these transformants, energized by incorporated cytochrome-c oxidase, showed 3-4-fold increased rates of arginine uptake compared to vesicles from wild-type cells. The KT values were 32.5 microM in vesicles from wild-type and 28.6 microM in vesicles from transformed cells; the corresponding in vivo values were 17.5 microM and 11.4 microM, respectively. It could be demonstrated that unidirectional arginine transport and accumulation also exist in vesicles; thus, unidirectional influx is not related to cellular compartmentation.

Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells were transformed with an S. pombe expression vector containing a full-length cDNA of the Chlorella hexose transporter. The transformed cells accumulated 3-O-methylglucose up to 10-fold, whereas wild-type S. pombe and control transformants could only equilibrate this sugar analogue. In a pH-jump experiment, in which extracellular pH was lowered by 1.9 units, the accumulation ratio was increased in transformed cells but not in control cells. This result indicates that the gene product, Chlorella H+/glucose-symporter protein, and a pH gradient suffice for active sugar uptake. Km values for glucose, 6-deoxyglucose, and 3-O-methylglucose of 1.5 x 10(-5) M, 2.7 x 10(-4) M, and 1.0 x 10(-3) M, respectively, were identical in Chlorella and in S. pombe cells transformed with Chlorella cDNA and approximately 100-fold lower than those of the endogenous transport system of S. pombe.