Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance.

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.

Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p.

The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid serine- and threonine-rich protein involved in flocculation. We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm. The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway. The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation. Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network. The membranous forms of Flo1p were shown to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle. The expression of truncated forms with the hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium. The hydrophobic C terminus, which is a putative GPI anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall. Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation. On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.

Colocalization of stomatin (band 7.2b) and actin microfilaments in UAC epithelial cells.

Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was investigated in a human epithelial cell line in which the expression of this protein is up-regulated after treatment with interleukin-6 and dexamethasone. A monoclonal antibody against stomatin was used to perform immunofluorescence and immunoelectron microscopy. The data show that stomatin concentrates preferentially in small plasma membrane protrusions. It is also found in abundance in a juxtanuclear structure possibly derived from the Golgi apparatus. Fluorescent double staining using the anti-stomatin antibody and the actin binding drug phalloidin shows a significant degree of colocalization of stomatin and cortical actin microfilaments. This association remains after actin filament disruption disruption by cytochalasin D treatment indicating a strong connection between stomatin and the membrane-associated cytoskeleton.

Genetic and biochemical characterization of the UGP1 gene encoding the UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae.

We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI [Purnelle B., Skala, J., Van Dijck, L. & Goffeau, A. (1992) Yeast 8, 977-986] encodes UDP-glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP. Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies. In particular, the amino acid sequence motifs involved in the binding of glucose 1-phosphate and UDP-Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi-copy expression of YKL248 resulted in a 40-fold increase in UGPase activity. This gene was, therefore, renamed UGP1. Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability. Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted. This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild-type gene under the control of the glucose-repressible galactose-inducible promoter. The effects of overproducing UGPase on the cell metabolism and morphology were carbon-source dependent. On glucose medium, the 40-fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP-Glc, with no significant effect on growth. In contrast, on galactose, the 40-fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3-5-fold increase in the concentrations of UDP-Glc, UDP-Gal and galactose 1-phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation. Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium. Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5-10% wild-type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium. However, the 10-fold reduction of UGPase activity induced a multi-budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell-wall beta-glucan content. All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP-Glc in yeast metabolism.

Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae.

The PMA2 gene is a presumed isogene of the PMA1 gene, encoding the major yeast plasma membrane H(+)-ATPase. When controlled by its own promoter, PMA2 in multiple copies does not complement a deficient PMA1 gene. Under the control of the PMA1 promoter, however, and expressed on a centromeric plasmid in yeast strains specially designed for stable expression, the PMA2 gene replaces the PMA1 gene to some extent, allowing growth on standard medium but not on acidic media. Plasma membranes of cells expressing only the PMA2 enzyme display low ATPase activity correlating with low amounts of PMA2 protein. This low activity is maintained throughout growth and does not increase when overexpression is favored by increased gene dosage. Immunoelectron microscopy reveals a dramatic proliferation of intracellular structures (probably endoplasmic reticulum) in which overexpressed PMA2 protein accumulates. Overexpression of PMA1 ATPase causes a similar phenomenon, but quantitative effects are lower compared to PMA2. These results indicate that the PMA2 gene encodes a functional plasma membrane H(+)-ATPase and suggest a specific control of the intracellular traffic of plasma membrane ATPase.

Properties of HIV membrane reconstituted from its recombinant gp160 envelope glycoprotein.

Human immunodeficiency virus (HIV) membrane has been reconstituted from the recombinant envelope glycoprotein precursor (gp160) by a detergent dialysis technique. Electron microscopy shows that gp160-virosomes are spherical vesicles with a mean diameter identical to that of viral particles. Enzyme-linked immunosorbent assay and immunogold labeling demonstrate efficient association of gp160 with lipid vesicles and proteolysis treatment reveals an asymmetric insertion with about 90% of glycoproteins having their gp120-moiety pointing outside. Glycoproteins are organized as dimers and tetramers and gp160 retains its ability to specifically bind CD4 receptor after reconstitution into virosome.

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.

Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma.

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles.

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.

Effect of suloctidil on rat liver.

The effect of suloctidil (120 mg/kg body weight PO for 3 weeks) on rat liver was investigated using biochemical and morphological methods: enzymatic activities characteristic of the main cellular compartments were used as biochemical markers of hepatocyte function and morphometry was applied to investigate morphological changes. No sign of hepatotoxicity was evidenced after suloctidil treatment (liver weight; cytochrome c oxidase; glucose 6-phosphatase; NADPH-cytochrome c reductase; D-amino acid oxidase; urate oxidase; fatty acid oxidation; peroxisomal number, volume and size distribution). Suloctidil increased catalase activity by 22% without morphologically detectable changes in the peroxisomes. After suloctidil treatment, slightly increased mitochondrial volume fraction and slightly decreased mitochondrial number were noted without significant changes in cytochrome c oxidase. Clofibrate, at the same dose, increased NADPH-cytochrome c reductase, catalase, acylCoA oxidase, mitochondrial and peroxisomal number and volume fraction, and decreased urate oxidase activity.

Hypertrophy of the rat Golgi complex during enhancement of cholesterol and bile acids synthesis.

There are some evidences that the Golgi apparatus could be involved in bile salts transport. In this study we investigate the morphology of the Golgi apparatus in rats with a chronic bile fistula or treated with 4% cholestyramine, a bile salts chelating agent. In both models liver histology was normal by light microscopy. With electron microscopy--compared to sham groups--the only obvious change was the enlargement of the Golgi complex. Morphometric data confirmed that the Golgi rich area volume density was almost doubled after chronic bile fistula or cholestyramine treatment. These data give another support for the participation of the Golgi complex in bile salts transport.

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes.

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 7. Size, shape and dynamism of secondary lysosomes.

Digestion of macromolecules inside secondary lysosomes results in small molecules leaving the lysosome by permeation. This may cause a drop in osmotic pressure and a consequent shrinkage of the lysosome. A membrane invagination may be formed which becomes internalized and then digested. This digestion causes new shrinkage. Minimal-sized lysosomes may ensue. The later are again engaged in digestive events when fusing with larger lysosomes. Through consecutive shrinkage and fusion cycles of many lysosomes residues slowly accumulate. Shrinkage may be counteracted by adding digestible material without increasing the lysosomal membrane surface. Transfer tubules can do so. They deliver their content to multivesicular bodies. The other lysosome types, however, are subject to shrinkage since they fuse with organelles (phagosomes, primary lysosomes) which insert at least part of their membrane.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 5. Autophagy.

ER, mature Golgi, plasma and lysosomal membranes are all able to sequester bits of cytosol and eventually some organelles. These four modes to provide in autophagy are complementary to each other. Apart from the digestion of the sequestered material, the implicated membranes (respectively ER, Golgi, plasma or lysosomal membranes) are partially degraded. This may counterbalance the continuous new formation of these membranes. It is proposed that the thin outer membrane of ER-derived autophagosomes is gradually converted into thick lysosomal membrane through consecutive fusions with different types of Golgi vesicles and with small dense bodies.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 6. Golgi apparatus.

Luminal materials from the ER are supplied to the Golgi apparatus (GA) either via precisternal elements (PE) detaching from the ER and building up a first cisterna, or via direct luminal continuities with the sER. Membrane supply is believed to follow only the first of these two pathways. It is further argued that during the processing of luminal materials and membrane - two Golgi activities that proceed at a different rate - bits of membrane carrying the differentiation enzymes are recycled to less differentiated membranes in the form of Golgi vesicles (GV).

A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.

A three-dimensional study of organelle interrelationships in regenerating rat liver. 4. Multivesicular bodies.

Double-membraned plasma-membrane-loops are formed from the plasma membrane and released into the cytoplasm. The vesiculation of mainly their inner membrane may transform them into MVB. The so-formed internal vesicles contain cytosol. In many MVB the surface of all their internal vesicles to gether corresponds well with the surface of their bordering membrane. This correlation may be the result of a dynamic equilibrium whereby degradation (and partial recyclage) of internal vesicles is compensated for by the formation of new internal vesicles. When this equilibrium becomes disturbed, MVB may show much less internal vesicles. Such 'depleted' MVB are often producing peribiliary vesicles and may themselves transform into lacunate emptying into the bile canaliculi.