GENETIC CONTROL OF MITOCHONDRIAL THYMIDINE KINASE IN HUMAN-MOUSE AND MONKEY-MOUSE SOMATIC CELL HYBRIDS

Distinctive thymidine (dT) kinase molecular forms are present in mouse, human, and monkey mitochondria. Disk polyacrylamide gel electrophoresis (disk PAGE) analyses have shown that the mitochondrial-specific dT kinases differ from cytosol dT kinases in relative electrophoretic mobilities (Rm). Furthermore, the mouse mitochondrial dT kinase differs in Rm value from primate mitochondrial dT kinases. The mouse and primate cytosol dT kinases can also be distinguished. Disk PAGE analyses have been carried out on the cytosol and mitochondrial dT kinases of human-mouse (WIL-8) and monkey-mouse (mK·CV^III) somatic cell hybrids in order to learn whether the mitochondria of the hybrid cells contained murine mitochondrial-specific, primate mitochondrial-specific, or both dT kinases. WIL-8 cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse fibro blasts and from cytosol dT kinase-positive, mitochondrial dT kinase-positive human embryonic lung cells; they contained mostly mouse chromosomes and a few human chromosomes, including the determinant for human cytosol dT kinase. The mK·CV^III cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse kidney cells and from cytosol dT kinase-positive, mitochondrial dT kinase-positive monkey kidney cells; they contained mostly mouse chromosomes and a few monkey chromosomes, including the determinant for monkey cytosol dT kinase. Disk PAGE analyses demonstrated that the mitochondria of human-mouse and monkey-mouse somatic cell hybrids contained the mouse-specific mitochondrial dT kinase but not the human- or monkey-specific mitochondrial dT kinase. These findings suggest that primate cytosol and mitochondrial thymidine kinase genes are coded on different chromosomes.     

Reconstitution of acid secretion in digitonin-permeabilized rabbit gastric glands. Identification of cytosolic regulatory factors

When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by [14C]aminopyrine accumulation, and they never recovered by supplement with cytosol prepared from gastric mucosa. However, the permeabilized glands elicited acid secretion when brain cytosol was supplemented. Fractionation of gastric cytosol by gel filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cytosol-stimulated acid secretion. Brain cytosol contained only the former stimulatory factor. With further gel filtration, the 30-kDa activator was separated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of which are necessary for full activity. We purified peak 1 from bovine brain, and phosphatidylinositol transfer protein (PITP) was identified as the main component of the activity. The stimulating activity in brain and gastric mucosa correlated with the contents of PITP, and recombinant PITP mimicked the effect of peak 1, suggesting that PITP is one of the essential components in gastric acid secretion. When gastric glands were stimulated, the inhibitory activity, but not stimulatory activity, in the cytosol was increased. This suggests a regulatory mechanism such as stimulation translocates the inhibitory component from the secretory site on the membrane to cytosol. These results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

Distinctive properties of mitochondrial thymidine(dT)kinase from bromodeoxyuridine(dBU)-resistant mouse lines

dBU-resistant mouse lines lack detectable dT kinase activity in the high speed supernatant (cytosol) cell fraction. However, they contain a mitochondrial dT kinase, which sediments more slowly in glycerol gradients than the cytosol enzyme of parental mouse lines, exhibits a disc PAGE mobility relative to the tracking dye (Rm) of about 0.7–0.8, and utilizes ATP, UTP, GTP, and CTP as phosphate donors. The mitochondrial fraction of parental cells also contains this 0.7–0.8 Rm activity and, in addition, a minor dT kinase activity which migrates faster than the cytosol enzyme, but utilizes only ATP as phosphate donor. The cytosol dT kinase of parental mouse lines exhibits an Rm of about 0.2–0.3 and utilizes only ATP as phosphate donor.     

A truncated form of the Pho80 cyclin of Saccharomyces cerevisiae induces expression of a small cytosolic factor which inhibits vacuole inheritance.

Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of a soluble inhibitory factor. When mixed with wild-type cytosol, vac5-1 cytosol inhibits the activity of wild-type cytosol. vac5-1 cytosol treated with trypsin or papain is still able to inhibit the activity of Aid-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein traction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-l small-molecule fraction retains the full ability to inhibit fusion. Thus, the vac5-1 allele of PHO80 induces the synthesis of a small molecule that is an inhibitor of vacuole inheritance.     

RhoGDIs revisited: novel roles in Rho regulation

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.     

Cytochrome c release and caspase activation during menadione-induced apoptosis in plants

We report here the detection of the release of cytochrome c from mitochondria into the cytosol during menadione-induced apoptosis in tobacco protoplasts. Western blot analysis indicated that the caspase specific inhibitors AC-DEVD-CHO (Ac-Asp-Glu-Val-Asp-aldehyde) and AC-YVAD-CHO (N-acetyl-Try-Val-Ala-aspartinal) inhibited the degradation of a caspase 3 specific substrate PARP (poly(ADP-ribose) polymerase), and they had no effect on the release of cytochrome c. Further study showed that menadione could not induce apoptosis of mouse liver nuclei in tobacco cytosol extract containing no mitochondria. However, when cytochrome c or mitochondria was added into the cytosol extract, apoptosis of mouse liver nuclei and the degradation of PARP could both be detected. The results provide strong evidence that menadione can induce apoptosis in tobacco protoplasts via the release of cytochrome c from mitochondria into the cytosol.     

Role of cytosol in the stimulation of RNA transport in vitro during cardiac hypertrophy in rats.

The 100,000 g supernatant isolated from hypertrophic hearts on fractionation by (NH4)2SO4 and DEAE-cellulose chromatography showed an enhanced RNA-transport activity when incubated with isolated nuclei from sham-operated hearts in vitro. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are enriched in the DEAE-cellulose fractions exhibiting maximal transport activity, and they are phosphorylatable. Pretreatment of the cytosol with antibodies to the Mr-68,000 and -32,000 proteins decreases the transport activity of the cytosol from 14% to 4.25%. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are translocated from the cytosol to the nuclear envelope under conditions of RNA transport in vitro. Our results here suggest that at least two of these proteins, those of Mr 68,000 and 32,000, play an indispensible role in the nucleocytoplasmic RNA transport in vitro. By making use of a specific myosin heavy-chain B-gene probe and hybridization, we have also shown the effect of cytosol on the transport of myosin heavy-chain mRNA from nucleus to cytosol.     

Cytosolic and nuclear aggregation of the amyloid beta-peptide following its expression in the endoplasmic reticulum

Misfolded secretory and membrane proteins are known to be exported from the endoplasmic reticulum (ER) to the cytosol where they are degraded by proteasomes. When the amount of exported misfolded proteins exceeds the capacity of this degradation mechanism the proteins accumulate in the form of pericentriolar aggregates called aggresomes. Here, we show that the amyloid beta-peptide (Abeta) forms cytosolic aggregates after its export from the ER. These aggregates share several constituents with aggresomes. However, Abeta aggregates are distinct from aggresomes in that they do not accumulate around the centrosome but are distributed randomly around the nucleus. In addition to these cytosolic aggregates, Abeta forms intranuclear aggregates which have as yet not been found for proteins exported from the ER. These findings show that proteins exported from the ER to the cytosol which escape degradation by the proteasome are not necessarily incorporated into aggresomes. We conclude that several distinct aggregation pathways may exist for proteins exported from the ER to the cytosol.     

Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits

As a final step in endocytosis, clathrin-coated pits must separate from the plasma membrane and move into the cytosol as a coated vesicle. Because these events involve minute movements that conventional light microscopy cannot resolve, they have not been observed directly and their dynamics remain unexplored. Here, we used evanescent field (EF) microscopy to observe single clathrin-coated pits or vesicles as they draw inwards from the plasma membrane and finally lose their coats. This inward movement occurred immediately after a brief burst of dynamin recruitment and was accompanied by transient actin assembly. Therefore, dynamin may provide the trigger and actin may provide the force for movement into the cytosol.     

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosol deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey, and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. These findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybrid cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed. The chick-mouse hybrid cells contained cytosol dCMP deaminase activity, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.This investigation was aided by Grant Q-163 from the Robert A. Welch Foundation and by USPHS Grants CA-06656-12 and 1-K6-AI 2352 from the National Cancer Institute and the National Institute of Allergy and Infectious Diseases.     

Size‐dependent secretory protein reflux into the cytosol in association with acute endoplasmic reticulum stress

Once secretory proteins have been targeted to the endoplasmic reticulum (ER) lumen, the proteins typically remain partitioned from the cytosol. If the secretory proteins misfold, they can be unfolded and retrotranslocated into the cytosol for destruction by the proteasome by ER‐Associated protein Degradation (ERAD). Here, we report that correctly folded and targeted luminal ER fluorescent protein reporters accumulate in the cytosol during acute misfolded secretory protein stress in yeast. Photoactivation fluorescence microscopy experiments reveal that luminal reporters already localized to the ER relocalize to the cytosol, even in the absence of essential ERAD machinery. We named this process “ER reflux.” Reflux appears to be regulated in a size‐dependent manner for reporters. Interestingly, prior heat shock stress also prevents ER stress‐induced reflux. Together, our findings establish a new ER stress‐regulated pathway for relocalization of small luminal secretory proteins into the cytosol, distinct from the ERAD and preemptive quality control pathways. Importantly, our results highlight the value of fully characterizing the cell biology of reporters and describe a simple modification to maintain luminal ER reporters in the ER during acute ER stress.     

Biochemically distinct vesicles from the endoplasmic reticulum fuse to form peroxisomes

As a rule, organelles in eukaryotic cells can derive only from pre-existing organelles. Peroxisomes are unique because they acquire their lipids and membrane proteins from the endoplasmic reticulum (ER), whereas they import their matrix proteins directly from the cytosol. We have discovered that peroxisomes are formed via heterotypic fusion of at least two biochemically distinct preperoxisomal vesicle pools that arise from the ER. These vesicles each carry half a peroxisomal translocon complex. Their fusion initiates assembly of the full peroxisomal translocon and subsequent uptake of enzymes from the cytosol. Our findings demonstrate a remarkable mechanism to maintain biochemical identity of organelles by transporting crucial components via different routes to their final destination.     

Identification of multiple protein kinases in normal human lymphocytes.

The protein kinase activity of a 10,000 g supernatant of purified human lymphocytes can be resolved by DEAE-cellulose chromatography into six protein kinase fractions: three of them phosphorylate casein preferentially, and three histones. The same procedure with the corresponding nuclear fraction yields only two casein kinases. All these fractions, except one casein kinase of the cytosol, have been studied with respect to protein and nucleotide specificity, effect of salts and of cyclic nucleotides, sedimentation, etc. The results obtained indicate that the enzyme fractions of the cytosol have distinct characteristics, suggesting that they are different protein kinases, and that the nuclear kinases are similar to the two main casein kinases of the cytosol.     

Cytoskeletal proteins of the cell surface in Tetrahymena : II. Turnover of major proteins

The turnover of surface membrane-associated cytoskeletal proteins has been studied in Tetrahymena. These proteins undergo rapid turnover and appear to be in equilibrium with precursor pools within the cytosol. Carefully controlled pulse-chase experiments, alone and in combination with cycloheximide, have shown that labeled proteins accumulated in the cytoskeleton after they were no longer being synthesized within the cell. The movement of subunits from the cytoskeleton to the cytosol was also demonstrated using tubulin isolated from these two compartments within the cell.     

Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)‐resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain‐of‐cytosolic functions—a phenomenon we name ER to Cytosol Signaling (ERCYS).     

Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum

The main reaction of N-glycosylation of proteins is the transfer 'en bloc' of the oligosaccharide moieties of lipid intermediates to an asparagine residue of the nascent protein. For the past 15 years, a few laboratories including ours have shown that the process was accompanied by the release of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn(1)) or two GlcNAc (OS-Gn(2)) at the reducing end. The aim of this review is to gather the evidence for the different origins of these soluble oligomannosides, to examine their subcellular location and intracellular trafficking. Furthermore, using Brefeldin A we demonstrated that this released oligomannoside material could be the substrate for the Golgi glycosidases and glycosyltransferases. Indeed, released oligomannoside never reach the Golgi vesicles either because they are directly produced in the cytosol as has been demonstrated for oligosaccharide-phosphates and for neutral oligosaccharides possessing one GlcNAc at the reducing end or because they are actively transported out of the rough endoplasmic reticulum to the cytosol. One of the functions of oligomannoside trafficking between rough endoplasmic reticulum, cytosol and lysosomes could be to prevent these oligosaccharides for competing with glycosylation in the Golgi.     

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.     

Membrane Bound GSK-3 Activates Wnt Signaling through Disheveled and Arrow

Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. Here we examine the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. We find that targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. Our results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol.