Effect of KEPI (Ppp1r14c) deletion on morphine analgesia and tolerance in mice of different genetic backgrounds: when a knockout is near a relevant quantitative trait locus.

We previously identified KEPI as a morphine-regulated gene using subtractive hybridization and differential display PCR. Upon phosphorylation by protein kinase C, KEPI becomes a powerful inhibitor of protein phosphatase 1. To gain insights into KEPI functions, we created KEPI knockout (KO) mice on mixed 129S6xC57BL/6 genetic backgrounds. KEPI maps onto mouse chromosome 10 close to the locus that contains the mu-opioid receptor (Oprm1) and provides a major quantitative trait locus for morphine effects. Analysis of single nucleotide polymorphisms in and near the Oprm1 locus identified a doubly-recombinant mouse with C57BL/6 markers within 1 Mb on either side of the KEPI deletion. This strategy minimized the amount of 129S6 DNA surrounding the transgene and documented the C57BL/6 origin of the Oprm1 gene in this founder and its offspring. Recombinant KEPIKO mice displayed (a) normal analgesic responses and normal locomotion after initial morphine treatments, (b) accelerated development of tolerance to analgesic effects of morphine, (c) elevated activity of protein phosphatase 1 in thalamus, (d) attenuated morphine reward as assessed by conditioned place preference. These data support roles for KEPI action in adaptive responses to repeated administration of morphine that include analgesic tolerance and drug reward.

The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.

Role of small G proteins in yeast cell polarization and wall biosynthesis.

In the vegetative (mitotic) cycle and during sexual conjugation, yeast cells display polarized growth, giving rise to a bud or to a mating projection, respectively. In both cases one can distinguish three steps in these processes: choice of a growth site, organization of the growth site, and actual growth and morphogenesis. In all three steps, small GTP-binding proteins (G proteins) and their regulators play essential signaling functions. For the choice of a bud site, Bud1, a small G protein, Bud2, a negative regulator of Bud1, and Bud5, an activator, are all required. If any of them is defective, the cell loses its ability to select a proper bud position and buds randomly. In the organization of the bud site or of the site in which a mating projection appears, Cdc42, its activator Cdc24, and its negative regulators play a fundamental role. In the absence of Cdc42 or Cdc24, the actin cytoskeleton does not become organized and budding does not take place. Finally, another small G protein, Rho1, is required for activity of beta (1-->3)glucan synthase, the enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall. In all of the above processes, G proteins can work as molecular switches because of their ability to shift between an active GTP-bound state and an inactive GDP-bound state.

Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin.

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.

Rho1p, a yeast protein at the interface between cell polarization and morphogenesis.

The enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall, beta(1-->3)-D-glucan synthase (also known as 1,3-beta-glucan synthase), requires a guanosine triphosphate (GTP) binding protein for activity. The GTP binding protein was identified as Rho1p. The rho1 mutants were defective in GTP stimulation of glucan synthase, and the defect was corrected by addition of purified or recombinant Rho1p. A protein missing in purified preparations from a rho1 strain was identified as Rho1p. Rho1p also regulates protein kinase C, which controls a mitogen-activated protein kinase cascade. Experiments with a dominant positive PKC1 gene showed that the two effects of Rho1p are independent of each other. The colocalization of Rho1p with actin patches at the site of bud emergence and the role of Rho1p in cell wall synthesis emphasize the importance of Rho1p in polarized growth and morphogenesis.

Accumulation of Golgi-specific mannosyltransferases in Candida albicans cells grown in the presence of brefeldin A.

The fungal metabolite brefeldin A (BFA) is known for its ability to block the secretory process in eukaryotic cells by interfering in the endoplasmic reticulum (ER) to Golgi membrane traffic, causing the disassembly of Golgi apparatus and redistribution of Golgi enzymes into the ER. In sensitive yeasts, underglycosylated forms of secretory proteins accumulate in the cytoplasm in the presence of BFA. We investigated whether the incomplete glycosylation of mannoproteins could be due to repression of the synthesis of Golgi-located terminal mannosyltransferases and whether the underglycosylated mannoproteins can be incorporated into the cell walls in Candida albicans. However, we found that the microsomal membranes isolated from the yeast cells grown in the presence of 14 micrograms.mL-1 of BFA had on average three times higher overall specific activity of mannan synthase than membranes from control cells. The increase in specific activity of mannan synthase was mainly due to accumulation of Golgi-specific mannosyltransferases responsible for elongation of the O-glycosidically linked mannooligosaccharides and for the synthesis of the N-glycosidically linked mannan outer chain. As a consequence, the mannans synthesized in vitro from GDP-[U-14C]mannose by the membranes from cells grown in the presence of BFA had longer O-glycosidically linked oligosaccharides and longer side-chains in the N-glycosidically linked polymannose part of the molecule than mannans synthesized by membranes from the control cells. Contrary to results obtained in vitro, the structural features of cell wall mannans isolated from intact BFA-grown and from control cells were almost indistinguishable.