The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells.

. Yeast mutants of cell cycle gene cdc481 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque . The gene was cloned and disruption proved it to be essential . The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein . Yeast Secl8p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Paslp,

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphosphatase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Sitedirected mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycledependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.

Trypanosoma brucei bloodstream forms express a densely packed surface coat consisting of identical variant surface glycoprotein (VSG) molecules. This surface coat is subject to antigenic variation by sequential expression of different VSG genes and thus enables the cells to escape the mammalian host's specific immune response. VSG turnover was investigated and compared with the antigen switching rate. Living cells were radiochemically labeled with either 125I-Bolton-Hunter reagent or 35S-methionine, and immunogold-surface labeled for electron microscopy studies. The fate of labeled VSG was studied during subsequent incubation or cultivation of labeled trypanosomes. Our data show that living cells slowly released VSG into the medium with a shedding rate of 2.2 +/- 0.6% h-1 (t1/2 = 33 +/- 9 h). In contrast, VSG degradation accounted for only 0.3 +/- 0.06% h-1 (t1/2 = 237 +/- 45 h) and followed the classical lysosomal pathway as judged by electron microscopy. Since VSG uptake by endocytosis was rather high, our data suggest that most of the endocytosed VSG was recycled to the surface membrane. These results indicate that shedding of VSG at a regular turnover rate is sufficient to remove the old VSG coat within one week, and no increase of the VSG turnover rate seems to be necessary during antigenic variation.