A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.     

Mr 46,000 mannose 6-phosphate receptor. The role of histidine and arginine residues for binding of ligand.

The chemical modification of histidine and arginine residues results in a loss of binding of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) to a phosphomannan affinity matrix (Stein, M., Meyer, J. E., Hasilik, A., and von Figura, K. (1987) Biol. Chem. Hoppe-Seyler 368, 927-936). Reversal of the modification or presence of mannose 6-phosphate during the modification partially restores or protects the binding activity, indicating that histidine and arginine residues contribute to the mannose 6-phosphate binding site. The 5 histidine and 8 arginine residues within the luminal domain of MPR 46, which contains the ligand binding site, were exchanged by site-directed mutagenesis. Only the conservative replacement of His-131 and Arg-137 by serine and lysine, respectively, results in a loss of binding activity without affecting other properties of the receptor such as the presence of intramolecular disulfide bonds, immunoreactivity, processing of N-linked oligosaccharides, formation of dimers, intracellular distribution, and surface expression. Conservative replacement of other histidine and arginine residues did not affect the binding activity. Nonconservative replacement of several arginine residues reduced binding activity and immunoreactivity, indicating that the loss of a positive charge at these positions alters the folding of MPR 46. We conclude from these results that His-131 and Arg-137 are essential for binding of ligands by MPR 46.     

The Function of Yeast Epsin and Ede1 Ubiquitin-Binding Domains During Receptor Internalization

The formation of a primary endocytic vesicle is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. Epsins and Eps15-like proteins are ubiquitin-binding proteins that act early in this process. The yeast epsins, Ent1 and Ent2, carry functional ubiquitin-interacting motifs (UIMs), whereas the yeast Eps15-like protein, Ede1, has a C-terminal ubiquitin-associated (UBA) domain. Analysis of mutants lacking early endocytic adaptors reveals that the ubiquitin-binding domains (UBDs) of Ent2 and Ede1 are likely to function primarily to mediate protein–protein interactions between components of the early endocytic machinery. Cells that lack epsin and Ede1 UBDs are able to internalize activated, ubiquitinated receptors. Furthermore, under conditions in which epsin UIMs are important for receptor internalization, receptors internalized via both ubiquitin-dependent and ubiquitin-independent signals require the UIMs, indicating that UIM function is not restricted to ubiquitinated receptors. Epsin UIMs share function with non-UBD protein–protein interaction motifs in Ent2 and Ede1, and the Ede1 UBA domain appears to negatively regulate interactions between endocytic proteins. Together, our results suggest that the ubiquitin-binding domains within the yeast epsin Ent2 and Ede1 are involved in the formation and regulation of the endocytic network.     

Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.     

Liposome-encapsulated oxymorphone hydrochloride provides prolonged relief of postsurgical visceral pain in rats

Adequate pain control is necessary for optimal postsurgical recovery and humane treatment of laboratory and companion animals. Opioid drugs are currently the most potent analgesic agents available in human and veterinary medicine. Long-acting formulations of opioid drugs confer several important advantages over standard pharmaceutical preparations, especially for use in animals. A long-acting formulation of oxymorphone hydrochloride was produced by encapsulation into liposomes. Liposome-encapsulated (LE) oxymorphone was tested in a rat model of visceral postoperative pain. Rats were given one subcutaneous injection of LE oxymorphone (1.2 or 1.6 mg/kg of body weight) or standard oxymorphone (0.3 mg/kg) at the time of intestinal transection or resection. A single administration of LE oxymorphone hydrochloride was as effective for relief of postoperative pain in rats (P = 0.18), as were multiple (q4 h or q8 h) injections of 0.3 mg/kg of the standard pharmaceutical preparation. The rats given LE oxymorphone prior to intestinal resection also had significantly higher body weight at three and seven days after surgery than did rats that were given standard oxymorphone. In conclusion, LE oxymorphone was effective in treating visceral pain associated with intestinal surgery in rats. On the basis of body weight gain, rats treated with LE oxymorphone had improved recovery outcome, compared with rats treated with repeated injections of standard oxymorphone.