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Calcium entry plays a major role in the induction of several forms of synaptic plasticity in different areas of the central nervous system. The spatiotemporal aspects of these calcium signals can determine the type of synaptic plasticity induced, e.g. LTP (long-term potentiation) or LTD (long-term depression). A vast amount of research has been conducted to identify the molecular and cellular signalling pathways underlying LTP and LTD, but many components remain to be identified. Calcium sensor proteins are thought to play an essential role in regulating the initial part of synaptic plasticity signalling pathways. However, there is still a significant gap in knowledge, and it is only recently that evidence for the importance of members of the NCS (neuronal calcium sensor) protein family has started to emerge. The present minireview aims to bring together evidence supporting a role for NCS proteins in plasticity, focusing on emerging roles of NCS-1 and hippocalcin.

The role of endocytosis in controlling a multitude of cell biological events is well established. Molecular and mechanistic characterization of endocytosis has predominantly focused on CME (clathrin-mediated endocytosis), although many other endocytic pathways have been described. It was recently shown that the BAR (Bin/amphiphysin/Rvs) and Rho GAP (GTPase-activating protein) domain-containing protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) is found on prevalent, pleiomorphic endocytic membranes, and is essential for the major, clathrin-independent endocytic pathway that these membranes mediate. This pathway is characterized by its ability to internalize GPI (glycosylphosphatidylinositol)-anchored proteins, bacterial toxins and large amounts of extracellular fluid. These membrane carriers are highly dynamic and associated with the activity of the small G-protein Cdc42 (cell division cycle 42). In the present paper, we review the role of GRAF1 in this CLIC (clathrin-independent carrier)/GEEC (GPI-anchored protein-enriched early endocytic compartment) endocytic pathway and discuss the current understanding regarding how this multidomain protein functions at the interface between membrane sculpting, small G-protein signalling and endocytosis.

The NHEJ (non-homologous end-joining) pathway is one of the major mechanisms for repairing DSBs (double-strand breaks) that occur in genomic DNA. In common with eukaryotic organisms, many prokaryotes possess a conserved NHEJ apparatus that is essential for the repair of DSBs arising in the stationary phase of the cell cycle. Although the bacterial NHEJ complex is much more minimal than its eukaryotic counterpart, both pathways share a number of common mechanistic features. The relative simplicity of the prokaryotic NHEJ complex makes it a tractable model system for investigating the cellular and molecular mechanisms of DSB repair. The present review describes recent advances in our understanding of prokaryotic end-joining, focusing primarily on biochemical, structural and cellular aspects of the mycobacterial NHEJ repair pathway.

Arf family GTP-binding proteins function in the regulation of membrane-trafficking events and in the maintenance of organelle structure. They act at membrane surfaces to modify lipid composition and to recruit coat proteins for the generation of transport vesicles. Arfs associate with membranes through insertion of an N-terminal myristoyl moiety in conjunction with an adjacent amphipathic alpha-helix, which embeds in the lipid bilayer when Arf1 is GTP-bound. In this issue of the Biochemical Journal, Lundmark et al. report that myristoylated Arfs in the presence of GTP bind to and cause tubulation of liposomes, and that GTP exchange on to Arfs is more efficient in the presence of liposomes of smaller diameter (increased curvature). These findings suggest that Arf protein activation and membrane interaction may initiate membrane curvature that will be enhanced further by coat proteins during vesicle formation.

Deletion and mutational analyses of the rat liver glycogen-targeting subunit (GL) of protein phosphatase 1 (PP1) have identified three separate domains that are responsible for binding of PP1, glycogen and phosphorylase a. The glycogen-binding domain spans the centre of GL between residues 144 and 231 and appears to be distinct from the glycogen-binding (storage) site of phosphorylase. The regulatory high-affinity binding site for phosphorylase a lies in the 16 amino acids at the C-terminus of GL. The PP1-binding domain is deduced to comprise the -RVXF- motif [Egloff, Johnson, Moorhead, Cohen and Barford (1997) EMBO J. 16, 1876–1887] located at residues 61–64 of GL and preceding lysine residues at positions 56, 57 and 59. A possible approach for increasing glycogen synthesis in certain disorders is discussed.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats and in adrenalectomized starved rats, and although this is known to be due to defective activation of glycogen synthase by glycogen synthase phosphatase, the underlying molecular mechanism has not been delineated. Glycogen synthase phosphatase comprises the catalytic subunit of protein phosphatase 1 (PP1) complexed with the hepatic glycogen-binding subunit, termed GL. In liver extracts of insulin-dependent diabetic and adrenalectomized starved rats, the level of GL was shown by immunoblotting to be substantially reduced compared with that in control extracts, whereas the level of PP1 catalytic subunit was not affected by these treatments. Insulin administration to diabetic rats restored the level of GL and prolonged administration raised it above the control levels, whereas re-feeding partially restored the GL level in adrenalectomized starved rats. The regulation of GL protein levels by insulin and starvation/feeding was shown to correlate with changes in the level of the GL mRNA, indicating that the long-term regulation of the hepatic glycogen-associated form of PP1 by insulin, and hence the activity of hepatic glycogen synthase, is predominantly mediated through changes in the level of the GL mRNA.

The reticulocyte 15-lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) is implicated in oxidative damage to reticulocyte mitochondria before their elimination by degradation during maturation to the erythrocyte. A proportion of the 15-lipoxygenase sediments with the mitochondrial-rich stromal fraction of density-gradient-fractionated rabbit reticulocytes suggesting a physical association with mitochondria before their elimination. Ca2+ promotes binding of reticulocyte 15-lipoxygenase to isolated rat liver and reticulocyte mitochondria and 15-lipoxygenase-mediated lipid peroxidation of mitochondrial lipids and free linoleic acid. Association of reticulocyte 15-lipoxygenase with isolated mitochondria is not simply a consequence of Ca(2+)-induced swelling, but implies that Ca2+ mediates translocation of soluble lipoxygenase to mitochondrial membranes. Therefore, Ca2+ may have an important physiological role in the regulation of 15-lipoxygenase-mediated targeting of reticulocyte mitochondria for degradation.

Antisera have been raised against two peptides from the sequence of D2 dopamine receptors: peptide 1 from the predicted second extracellular loop and peptide 2 from the predicted third intracellular loop. The antisera recognize specifically a 95 kDa band in Western blots of several bovine brain regions, which corresponds to the denatured D2 dopamine receptor, whereas in recombinant CHO cells expressing D2 dopamine receptors a 80 kDa band is seen. The antisera immunoprecipitate 10-20% of the D2 dopamine receptors from soluble preparations of bovine brain. The antisera recognize D2 dopamine receptors in immunofluorescence analyses of recombinant CHO cells bearing the receptor gene. The antisera directed against the third intracellular loop, but not those against the second extracellular loop, will interfere with the coupling of D2 dopamine receptors and G-proteins in bovine brain preparations.

Homologous cytosol was introduced into 3T3-L1 cells by two different methods. Erythrocytes loaded with radiolabelled cytosolic proteins extracted from 3T3-L1 cells were fused with the aid of Sendai virus to 3T3-L1 cells, which were then seeded to confluent and non-confluent cultures. Cytosolic proteins were also introduced into cells by the technique of scrape-loading. In confluent cells, injected cytosolic proteins were recovered largely (54-93%) in a sedimentable (6 X 10(6) gav.-min) fraction from recipient cells irrespective of the method of introduction or of radiolabelling of the injected proteins [(125I]iodination, reductive methylation with NaB3H4 and backbone labelling with L-[4,5-3H]leucine). The degradation of microinjected cytosolic proteins was in all cases inhibited by the lysosomotropic agent NH4Cl to a greater extent (32-75%) than that observed for endogenous cytosolic (less than or equal to 19%) proteins (labelled with L-[4,5-3H]leucine). In growing cells both endogenous total cell proteins and microinjected proteins were degraded at a slower rate than in confluent cell monolayers. The inhibition by NH4Cl of the degradation of both the endogenous and microinjected proteins is decreased compared with the inhibition observed in confluent monolayers. The results are discussed in terms of the cytoplasmic capacity to segregate microinjected homologous proteins before protein degradation can take place.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

n.d. properties of the four cathepsins are recorded in Table 1. Cathepsin S from rabbit spleen was similar to the previously described bovine cathepsin S except that it exhibited a different response in the two azocasein assays. As reported previously (Etherington, 1980), cathepsin N caused little degradation of azocasein. However, in the presence of 3M- urea, which is now used routinely in the assay for cathepsin L, cathepsin N exhibited substantial activity. Furthermore cathepsin N exhibited only a weak gelatinase activity unless 3M-urea was also included. Cathepsins N and S were com- pletely resolved from cathepsins B and L by IEF. IEF gels were prepared and the enzymes located by applying an agarose-substrate overlay. In each overlay examined the differences in specificity were confirmed from the intensities of the digestion zones. IEF gels were also used in an imniunodiffusion assay with an antiserum raised against rabbit cathepsin L (Etherington et al., 1984). A precipitin arc was formed only at the position of cathepsin L and not the positions of cathepsin B, N or S. This is the first time that all four cathepsins have been identified in a single tissue. It is concluded that they are different enzymes, but their respective contributions to intracellular proteolysis are still unclear.

of myosin light chain phosphorylation (e.g. England, 1983). In contrast, we have observed an increase in the phosphorylation of myosin light chain in response to both SK&F 94 120 and isoprenaline. Interestingly, it appeared that SK&F 94 120 caused a relatively greater phosphorylation of myosin light chain. These data are consistent with SK&F 94120 exerting its inotropic effects in guinea-pig hcart by inhibition of phosphodiesterase, and the subsequent increase in the levels of cyclic AMP-dependent protein phosphorylation. Although qualitatively the phosphorylation patterns were similar for both SK&F 94 120 and isoprenaline. quantitative differences were observed, particularly in the phosphorylation of myosin light chain. England, P. J . (1976) Hiochrm. J . 160 .295304 England, P. J . ( 198.7) in Cordicrc Metdwlisrn (Drake-Holland, A. J.

in the denser lysosome fractions of cells treated with E-64 is due to an increase in ubiquitin conjugates in these fractions. Ubiquitin has been suggested to be involved in the marking of proteins for non-lysosomal degradation [4]. Our results suggest that ubiquitination and/or aggregation may: ( a ) serve to ‘isolate’ polypeptides whose degradation by the lysosomal system has been prevented; ( 6 ) ‘mark’ polypeptides for lysosomal degradation; ( c ) be a feature of some normally ubiquitinated proteins, e.g. cell surface proteins [5 , 6, 71 which are degraded lysosomally and accumulate in the lysosome when lysosomal proteolysis is blocked. 3. Hershko, A,, Eytan, E. & Ciechanover, A. & Haas, A. L. (1982) J. Biol. Chem. 251,13964-13970 4. Ciechanover, A,, Finley, D. & Varshavsky, A. ( 1 984) J. Cell. Biochem. 24,27-58 5. Leung, D. W., Spencer, S. A,, Cachianes, G., Hammonds, R. G., Collins, C., Henzel, W. J., Barnard, R., Waters, M. J. & Wood, W. I. (1987) Nulure( London) 330,537-543 6. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Tremble, D. P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Fried, V. A,, Ullrich, A. & Williams, L. T. (1986) Nature(London) 323,226-232 7. Meyer, E. M., West, C. M. & Chau, V. (1986) J. Biol. Chem. 261, 14365-14368