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The role of phosphagens in regulation of intracellular Pi levels is likely to have evolved early. Thermodynamic poise of the phosphagen kinase reaction greatly impacts it. the phosphagens differ in certain physical properties, among them intrinsic diffusivity. The capacity for intracellular targeting of CK can be hypothesized to have evolved early as a way to facilitate energy transport in highly polarized cells. It would have been exploited for temporal ATP buffering and dynamic roles in metabolic regulation in cells with high, variable rates of aerobic energy production later.

Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two. Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretory pathway by largely unknown mechanisms. Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins. We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms. Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation. These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.

The Escherichia coli sigma(super.E)-dependent extracytoplasmic stress response is discussed. The response is controlled by the regulated proteolysis of an anti-sigma factor. The sigma(super.E) activity is determined largely by the ratio of RseA to sigma(super.E). RseA degrades quickly responding to extracytoplasmic stress. That leads to an increase in the free pool of sigma(super.E) and the stress response is set up.

African trypanosomes compartmentalize glycolysis in a microbody, the glycosome. When growing in the mammalian bloodstream, trypanosomes contain only a rudimentary mitochondrion, and the first seven glycolytic enzymes, including phosphoglycerate kinase, are located in the glycosome. Procyclic trypanosomes, growing in the gut of tsetse flies, possess a fully developed mitochondrion that is active in oxidative phosphorylation. The first six glycolytic enzymes are still glycosomal, but phosphoglycerate kinase is now found in the cytosol. We demonstrate here that bloodstream trypanosomes are killed by expression of cytosolic phosphoglycerate kinase. The toxicity depends on both enzyme activity and cytosolic location. One possible explanation is that cytosolic phosphoglycerate kinase creates an ATP-generating shunt in the cytosol, thus preventing full ATP regeneration in the glycosome and ultimately inhibiting the first, ATP-consuming, steps of glycolysis.

Immunoreactive and immunofluorescence studies using monoclonal antibodies directed against clathrin and gamma-adaptin, a complex containing a clathrin adaptor, as well as Western blot analyses were carried out to demonstrate the presence of clathrin and clathrin adaptors on tubulovesicles from mammalian oxyntic cells. Further, gamma-adaptin was co-purified with hydrogen-potassium adenosine triphosphatase indicating that tubulovesicles play a role in the trafficking of this transporter.

The effects of phorbol esters, epidermal growth factor (EGF) and angiotensin (ANG) II on the translocation of the alpha, delta and epsilon isozymes of protein kinase C in rat liver epithelial cells were investigated through Western blot analysis following subcellular fractionation of treated tissues. Phorbol esters induced the highest degree of membrane translocation of the different isozymes. Although the magnitude of translocation induced by both EGF and ANG II is low, the significant translocation of the delta and epsilon isozymes is enough to explain spatially defined effects.

Research was conducted to investigate the endocytic pathways of both a mouse and a human B cell line using quantitative immunoelectron microscopy on cryosections of cells pulse-chased with transferrin-HRP or BSA-gold as endocytic tracers. A maximum of six endocytic subcompartments were identified including an early MIIC type which indicates that it serves as an important entrance to the endocytic pathway for newly synthesized major histocompatibility complex (MHC) class II. Results indicate that B cells use conventional endocytic compartments to accomplish MHC class II presentation.

Analysis of cell affinities in the developing wing of Drosophila shows a division into the anterior (A) and posterior compartments (P), mediated by the transcription factor Engrailed (En) in P cells. Under control of En, P cells secrete the signalling protein Hedgehog (Hh), to which they cannot respond. It is hypothesized that the failure of A and P cells to intermix is caused by the Hh secreted by the P cells. A mutation of the gene smoothened is used to show that the affinity boundary that segregates the A and P cells is mainly a consequence of local Hh signalling.

Fractionation analysis on various protein targeting mutants was employed to determine whether or not the yeast vacuolar protein aminopeptidase I (API) utilizes a vesicular mechanism for vacuolar delivery. Results reveal that API is transported inside a double-membraned vesicle that is formed in the cytosol. Furthermore, it is found that two yeast mutants trap the API precursor inside a vesicular compartment. One of these mutant strains is essential in purifying an API transport intermediate.

A study was conducted on the influence of cAMP subcellular compartmentation in the beta-adrenergic stimulation of cardiac L-type calcium current. A technique based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion was used. Results indicated that cAMP compartmentation provides a general method through which cells discriminate among various external stimuli acting via a common signaling pathway.

Molecular cloning of an operon, cotJ, one of the 13 coat proteins which when inactivated induces sporulation with an altered pattern of coat polypeptides in Bacillus subtilis, revealed a tricistronic operon preceded by a strong sigmaE-like promoter. DNA mapping showed that expression of cotJ is completely dependent on spoIIGB, a DNA binding protein that encodes sigmaE, and that its precedes expression of cotA, but not cotE. This suggests that cotJ is involved in the early stage of coat assembly in B. subtilis.

Immunofluorescence tests in Bacillus subtilis were used to visualize cell-specific gene expression and subcellular protein localization. Cells treated with propidium iodide and with fluorescein-conjugated antibodies indicated that gene expression is exclusive to the mother cell compartment of sporangia at the septation and engulfment stages of morphogenesis when under the control of RNA polymerase sigmaE. A high level of compartmentalization was also observed in gene expression of cells under the control of sigmaG.

The identification of an epithelial cell-specific small GTPase is reported. The GTPase is called rab17, a member of the rab subfamily of small GTPases which have an important role in the regulation of membrane traffic in eukaryotic cells. The presence of distinct apical and basolateral regions in epithelial cells implies separate regulation of their transport pathways, and thus the existence of specific rab proteins. Northern blot, immunofluorescence and immunoelectron analyses showed that rab17 is present only in epithelial cells.

The zeta chains of T-cell antigen receptors (TCRs) undergo phosphorylation on tyrosine residues after TCR crosslinking. To determine the role of phosphorylated zeta proteins in TCR transport and function in CD4+CD8+ thymocytes, the subcellular compartment in which phosphorylated zeta (P-zeta) chains are located was determined. The results showed that P-zeta chains are entirely associated with mature TCR complexes that have been transported to the trans-Golgi complex or beyond. Therefore, P-zeta is not involved in the retention of the proteins in the endoplasmic reticulum.

The effects of caffeine and temperature on the distribution and organization of Golgi complex membranes were investigated. The results showed that at 20 degrees celsius, caffeine can effect the translocation of pre- and cis-Golgi compartment markers to the cell periphery. In contrast, Golgi stack markers remain perinuclear under the same conditions. These results indicate that caffeine and reduced temperature can be used to analyze the interface between the cis-Golgi and the Golgi stack.

The posterior-tail domain-specific integral plasma membrane glycoprotein CE9 of the rat testicular spermatozoon was characterized and its compartmentalization studied. It was found to be a Type Ia transmembrane protein identical to the MRC OX-47 T-cell activation antigen. In addition, it has a smaller molecular mass due to under-utilization of sites for asparagine-linked glycosylation. Moreover, its lateral diffusion within the posterior-tail plasma membrane domain of the testicular spermatozoon was measured and the conditions that support the redistribution of CE9 on spermatozoa were identified.

The mechanism by which cytoplasmic H+ - K+ - ATPase-bearing tubulovesicles fuse with the apical portion of gastric parietal cells during stimulation of HCl secretion by parietal cells was investigated. The study protocol involved searching for cytoskeletal proteins that associate with the gastric ATPase and stabilize its insertion in the parietal cell apical membrane. The results showed that ankyrin mediates the association of the gastric ATPase with spectrin and actin. These protein associations contribute to the maintenance of the polarized distribution of the gastric ATPase to the apical membrane during HCl secretion.

The Xenopus Vg1 mRNA is a transforming growth factor which remains localized to the vegetal cortex of late-stage oocytes until after gastrulation. An investigation was conducted to determine the cis- and trans-acting factors involved in Vg1 mRNA vegetal cortex localization. Competition experiments identified two protein binding regions in the 3' untranslated region (UTR) which are involved in vegetal localization. In addition, a 69-kDa protein which binds to this region was identified. These results suggest the involvement of the 3' UTR signals and cellular factors which recognize these signals in mRNA oocyte localization.

The distribution of various carbonic anhydrases (CA) within the the male rat hepatocyte was determined by analyzing various fraction preparations of the cells. The results showed that a CA type IV (CA IV) was associated with the microsomal membrane, specifically with the endoplasmic reticulum. However, this membrane-bound CA is only functionally accessible from the inside. In addition, CA II and CA III were detected in the cytosol.

The sorting of 5' nucleotidase (5NT) to the apical domain of the hepatocyte plasma membrane (PM) investigated. 5NT is an endogenous glycophosphatidylinositol (GPI)-anchored proteins in hepatocytes. Studies have shown that apical proteins are first sorted to the PM basolateral domain, endocytosed and then sorted to the apical domain. Using a protocol that resolves the apical or basolateral origin of PM vesicles, the study showed that 5NT is also sorted via the basolateral domain. However, transcytosis to the apical domain occurs slowly because GPI-linked proteins are endocytosed via a slower pathway independent of clathrin-coated pits.

Three methods for indirect visualization of thesubcellular distribution of dystrophin on skeletal muscle fiber membrane were developed. Dystrophin is the protein implicated in Duchenne muscular dystrophy.The study protocols involved incubation of single-teased muscle fibers with dystrophin antibodies, mechanical skinning of single-teased fibers and semithincryosectioning of human muscle fibers. The results showed that dystrophin molecules are not uniformly distributed in the skeletal muscle fiber membrane.

Thedifferential localization of the Acanthamoeba myosin I isoforms were investigated. The study protocol involved immunofluorescence and immunoelectronmicroscopy of vegetative and phagocytosing cells and determination of the totalcell contents of the three myosin isoforms by immunoprecipitation. The results indicated that myosin IA has a role in cytoplasmic vesicle transport and cortical contraction and myosin IB has a role in phagocytosis and extension of pseudopods, while myosin IC mediates contractile vacuole function.

The mechanism for targeting of the type I transmembrane carboxypeptidase Kex1p to the Golgi aparatus was investigated. Kex1p is involved in precursor processing in the yeast secretory pathway. The study protocol involved the examination of the cellular location of a series ofcarboxy-terminal truncations of the carboxypeptidase. The results showed that the cytoplasmically exposed carboxy-terminal is required for retention of the carboxipeptidase in the Golgi apparatus. Deletion of the carboxy-terminal retention signal causes default localization to the vacuolar membrane.

The subcellular localizationof Cu,Zn-superoxide dismutase (SOD) was determined by light and electron microscopic immunocytochemistry. The results showed that Cu,Zn-SOD from a variety of tissues, including human lung, baboon lung and rat liver, is diffusely located in the nucleus, the cell cytosol and in the peroxisomes. Thisis in contrast to earlier findings which localized Cu,Zn-SOD exclusively in theperoxisomes. The previous results may have arisen from weak fixation methods.

The biosynthesis of the Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), was investigated to understand how API is localized to the vacuole. API is a soluble hydrolase in the vacuole which does not have a standard signal sequence and has an unusual NH2-terminal propeptide. The results showed that the API precursor does not undergo glycosylation and that the removal of its propeptide is sec-independent. The API precursor does not enter the secretory pathway but remains in the cytoplasm and directly translocates to the vacuole via a unique mechanism different from that of other vacuolar proteins.

The targeting signals of vacuole-resident dipeptidyl aminopeptidase-B (DPAP-B) and the Golgi-resident DPAP-A were investigated. Both DPAP-B and DPAP-A are integral membrane proteins in yeast. Removal or replacement of the cytoplasmic, transmembrane or lumenal domain of DPAP-B did not affect its transport to the vacuole. On the other hand, overproduction of DPAP-A or deletion of a portion of its cytoplasmic domain caused its localization in the vacuole rather than in the Golgi apparatus. The results indicate that the vacuole is the default compartment for membrane proteins.

The relative distribution of acetylcholine receptor alpha-subunit (AChRa) and acetylcholine esterase (AChE) mRNAs was studied to determine whether protein synthesis in multinucleate cells is compartmentalized according to specific protein products. The results using in situ hybridization of chick cultured muscle cells showed that the two proteins differed in terms of the subpopulations of nuclei involved in mRNA synthesis and in the cytoplasmic distribution of these mRNAs, although some overlap was observed. These results suggest that protein groups with a common final location may be processed in the same cellular compartment.

The interaction of the Golgi apparatus (GA) with the microtubule apparatus was observed in semi-intact cells to gain insight into the mechanism for localization of the GA within a specific cell compartment at different cell cycle stages. The results showed that GA can enter semi-intact cells and associate stably with microtubules in a process termed 'Golgi capture' (GC). GC is dependent on ATP hydrolysis, physiological temperature and the presence of sufficient cytoplasmic dynein. Cytoplasmic dynein is mainly responsible for the subcellular localization of the GA.

Three classes of Purkinje cells were identified by immunostaining of a cerebellar section with both P-path and Zebrin II. These consist of two complementary groups distinctly immunoreactive to one antibody but not the other and a third group that contains double- labeled cells. No Purkinje cells could be detected that were unreactive to either antibody. The compartmentation of the cerebellum into developmental genetic modules may be responsible for the specific and reproducible topographyof the Purkinje cells.

Many enzymes of animals, especially enzymes that regulate metabolic pathways, exhibit one or more types of inter- and intracellular compartmentation. A given type of catalytic activity is effected by different isoforms (isozymes) of a class of enzyme in different tissues, cells, or organelles, and many enzymes alter their locations within the cell under different physiological conditions. These compartmentation properties reflect major features of animal organization and function, e.g., the occurrence of different metabolic tasks in different tissues or cellular compartments, and enormous changes in muscle metabolic rate during intense locomotion. Enzymes of glycolysis, notably phosphofructokinase (PFK) and hexokinase (HK), manifest these forms of compartmentation. PFK occurs in several isozyme forms that differ in kinetic properties. Skeletal muscle PFK binds reversibly to actin in a pH-dependent manner. Binding of PFK to actin affects the enzyme's catalytic activity, sensitivity to regulatory modulators, and structural stability in the face of decreasing intracellular pH ([pH.sub.i]). Eucaryotic PFKs arose from a gene duplication/fusion event of an ancestral procaryotic PFK. This increase in subunit mass from 36 kDa to 82 kDa in animal PFKs facilitated the evolution of additional regulatory sites and, we hypothesize, an actin binding capacity which varies among PFK isoforms. In vertebrates, hexokinase occurs in several isoforms that differ in kinetic properties and ability to bind to the outer surface of the mitochondrion. Binding of HK to mitochondria plays an important role in regulating glucose metabolism in glucose-dependent tissues like brain. The different abilities of vertebrate HK isozymes to bind to mitochondria are due to charge differences on the isozymes' surfaces. Invertebrate HKs are smaller than vertebrate HKs (ca. 50 kDa vs. 100 kDa), and cannot bind to mitochondria. The evolution of multiple isozyme forms of animal enzymes, and the evolution of capacities for reversible intracellular compartmentation through increases in protein size, are discussed in the context of the large genome sizes characteristic of most animals.