C201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars C201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/ Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association.

We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3+, which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope‐tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule‐associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin‐based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4‐64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild‐type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi‐to‐ER recycling in wild‐type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild‐type strain. We conclude that Klp3 plays a role in BFA‐induced membrane transport. The nucleotide sequence of S. pombe klp3+ was submitted to GenBank under Accession No. AF154055. Copyright © 2000 John Wiley & Sons, Ltd.

phosphorylates GskA and this activates it (Kim et al., 1999;Kim et al., 2002). The activity of ZAK1 is developmentally regulated, with kinetics that are approximately coincident with the activation profile of GskA. Furthermore, there is no peak of GskA kinase activity in a zakA-null strain (Kim et al., 1999).Glycogen synthase kinase 3 (GSK3) is a central regulator of metazoan development and the Dictyostelium GSK3 homologue, GskA, also controls cellular differentiation. The originally derived gskA-null mutant exhibits a severe pattern formation defect. It forms very large numbers of pre-basal disc cells at the expense of the prespore population. This defect arises early during multicellular development, making it impossible to examine later functions of GskA. We report the analysis of a gskA-null mutant, generated in a different parental strain, that proceeds through development to form mature fruiting bodies. In this strain, Ax2/gskA-, early development is accelerated and slug migration greatly curtailed. In a monolayer assay of stalk cell formation, the Ax2/gskAstrain is hypersensitive to the stalk cell-inducing action of DIF-1 but largely refractory to the repressive effect exerted by extracellular cAMP. During normal development, apically situated prestalk cells express the ecmB gene just as they commit themselves to stalk cell differentiation. In the Ax2/gskA-mutant, ecmB is expressed throughout the prestalk region of the slug, suggesting that GskA forms part of the repressive signalling pathway that prevents premature commitment to stalk cell differentiation. GskA may also play an inductive developmental role, because microarray analysis identifies a large gene family, the 2C family, that require gskA for optimal expression. These observations show that GskA functions throughout Dictyostelium development, to regulate several key aspects of cellular patterning.

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Background:A. gambiae transglutaminase AgTG3 cross-links Plugin within seminal fluids. Results: AgTG3 and Plugin were purified and their structure and activity characterized in vitro. Conclusion: AgTG3 forms a dimer in solution, analogous to human FXIII. Plugin is nonglobular in solution. AgTG3 is Ca 2ϩ -dependent and prefers Plugin as a substrate. Significance: Inhibition of AgTG3 cross-linking of Plugin is a possible method to chemosterilize male Anopheles mosquitoes.

Automated tritium-hydrogen exchange measurements have been made on the linear octapeptide Val5-angiotensin II amide. All Much has been written recently about the importance of polypeptide conformation, the relation conformation bears to biological activity, and about methods to determine the conformation in aqueous solution. In spite of this only a few examples can be found in the literature where the total conformation has been reliably established, even with cyclic polypeptides in which conformational possibilities are greatly restricted. In addition, present methods have been inadequate to cope with the problems of defining the conformation of small linear polypeptides devoid of covalent crosslinks. Here, the question may be raised as to whether or not there is a unique conformation, and indeed such peptides have been frequently referred to as "random coil" types. While the synthetic polyamino acids have been much studied as linear models, showing conformation in certain solvent environments, they represent special cases and are heterogenous with respect to size. With recent advances in synthetic capability for polypeptides, together with improved methods to assess the degree of randomness by combinations of techniques such as nuclear magnetic resonance (NMR), tritium-hydrogen exchange, circular dichroism, thin-film dialysis, etc., evidence is accumulating that certain of the linear polypeptides can have a unique conformation in a given aqueous solution, or at least exhibit a highly restricted degree of randomness. Such information is essential to an understanding of the molecular basis of action of polypeptide hormones.The linear octapeptides of angiotensin II are good models for conformational studies, as well as being of importance in their own right. Evidence was presented some years ago from thinfilm dialysis (1) and optical rotatory dispersion (ORD) measurements (2) indicating the presence of a unique and compact conformation of Val5-angiotensin II in certain aqueous environments. While another early study (3) that used ORD, titration, and hydrogen exchange was not interpreted in terms of secondary structure, all subsequent studies have been. For example, a structural change caused by high pH was first indicated by biological activity studies (4), and was supported by thin-film dialysis studies (5) and ORD measurements (6). Later, the possibility of two interchangeable forms was supported by thin-film dialysis and gel filtration studies (7). More recently, a crystalline form of angiotensin II has been reported (8).Evidence from automated tritium-hydrogen exchange presented in this paper strongly supports the interpretation of secondary structure in angiotensin II.

BackgroundIn humans, mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs.Methodology/Principal FindingsWe have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous GarsC201R/+ mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the GarsC201R/+ mice to two other mutants: the TgSOD1G93A model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1Loa) which has a defect in the heavy chain of the dynein complex. We found the Dync1h1Loa/+;GarsC201R/+ double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the GarsC201R mutation significantly delayed disease onset in the SOD1G93A;GarsC201R/+ double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated.Conclusions/SignificanceThese findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.