A new class of Paramecium surface proteins anchored in the plasma membrane by a glycosylinositol phospholipid. Membrane anchor of Paramecium cross-reacting glycoproteins.

Treatment of paramecia with ethanol or Triton X-100 solubilizes a major membrane protein, namely the surface antigen (SAg), and a set of glycopeptides in the range 40-60 kDa, which cross-react with the SAg. We demonstrate that these glycopeptides, called 'cross-reacting glycoproteins' (CRGs), are distinct molecules from the SAg. First, after purification of CRGs from ethanolic extracts of Paramecium primaurelia expressing the 156G SAg, the amino acid composition of a given CRG was found to be different from, and incompatible with, that of the 156G SAg. Secondly, we showed that the CRGs, although not immunologically detectable, are present in fractions containing the myristoylated form of the 156G SAg. The treatment of these fractions by phosphatidylinositol-specific phospholipases C enables us to reveal the CRGs through the unmasking of two distinct epitopes. One is the 'cross-reacting determinant' (CRD), initially described for the variant surface glycoproteins (VSGs) of Trypanosoma; the other determinant, called 'det-2355', is specific to the SAg and to the CRGs. Our results suggest that (1) phosphatidylinositol is covalently linked to the CRGs and (2) the CRD and the det-2355 are localized in the same region of the CRGs. We propose that the CRGs are a new set of surface proteins anchored in the cell membrane of Paramecium via a glycosylinositol phospholipid, in the same way as the SAgs.     

Purification of the temperature-specific surface antigen of Paramecium primaurelia with its glycosyl-phosphatidylinositol membrane anchor

The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.     

Specific cell-induced transformation of Paramecium surface antigens.

Paramecia may induce in other paramecia specific changes in the expression of genes determining surface protein synthesis, probably via cell-to-cell contact. Transformation from one antigenic type to a newly induced one is stimulated by the pretreatment of a cell with 5-fluorouracil. These observations are considered in terms of the regulation of the activity of genes in stable cells and in unstable cells which tend to transform spontaneously.     

Immunological evidence of a common structure between Paramecium surface antigens and Trypanosoma variant surface glycoproteins

The surface antigens (SAgs) of Paramecium and the variant surface antigens (VSGs) of Trypanosoma can be purified in two distinct molecular forms: a soluble form (solubilized in dilute ethanolic solution in the case of Paramecium, or in water for Trypanosoma) and a membranal form, amphiphile (solubilized in SDS). In trypanosomes, the enzymatic conversion of the membrane form into the soluble form is accompanied by the unmasking of a particular immunological determinant, called cross-reacting determinant (CRD), which is located in the COOH-terminal phospho-ethanolamine glycopeptide. We demonstrate immunological homologies between Paramecium SAgs and Trypanosoma VSGs. A determinant corresponding to the CRD of VSGs is borne by the ethanol-soluble form of the SAgs and by two cross-reacting light chains also present in ethanolic cellular extracts (together with the soluble form), and not by the membranal form of SAgs. Furthermore, we show that the membranal form of Paramecium SAgs can be converted into soluble form and that this enzymatic conversion also yields cross-reacting light chains. We also demonstrate that the membranal form is the physiological form in paramecia stably expressing a given SAg.     

Structural comparisons between the soluble and the GPI-anchored forms of the Paramecium temperature-specific 156G surface antigen.

Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra.     

Analysis of the conserved cysteine periodicity of Paramecium variable surface antigens

The major surface antigens expressed by free-living and parasitic protozoa commonly contain repeating cysteine motifs. Despite the common occurrence of these repeats their functional significance remains largely unexplored. In this paper we investigate the conserved cysteine repeats within the variable surface antigens of Paramecium tetraurelia. We show that deletion of 2 entire repeating units or portions of repeats near the N-terminus does not prevent expression of the A51 variable surface antigen. Alteration of a single cysteine to serine residue also has no effect on A51 expression. In contrast, deletions near the C-terminus of the protein have identified a small segment within the repeats that is required for expression on the surface. The required region contains a number of conserved amino acid residues, yet site-directed mutagenesis of two residues (serine and threonine to alanine) did not prevent expression. These studies demonstrate the feasibility of using deletion analysis to identify regions critical for the expression of cysteine-rich surface antigens. The relationship of these results to the structure and expression of cysteine-rich surface proteins in other protozoa is discussed.     

The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.     

Isolation and translation of mRNA coding for the variant surface antigens of Paramecium.

In the poly(A)+RNA isolated from the ciliate Paramecium primaurelia is found a discrete and abundant mRNA species of high molecular weight (corresponding to about 9,000 nucleotides). This mRNA species has size and abundance characteristics that identify it tentatively as the message coding for the variant cell-surface antigens. After microinjection of the high molecular weight mRNA into amphibian oocytes, polypeptides are synthesized that are immunoprecipitated specifically with antibodies directed against the homologous Paramecium antigen. On collecting the culture medium of oocytes microinjected with Paramecium mRNA, newly-synthesized complete antigen molecules (Mr approximately 300,000) can be recovered by immunoprecipitation.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

The Immobilization Antigen of Paramecium aurelia is a Single Polypeptide Chain

The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethanol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (∼250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.     

Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia.

The DNA sequences of the entire coding regions of the A and C type variable surface protein genes from Paramecium tetraurelia, stock 51 have been determined. The 8151 nucleotide open reading frame of the A gene contains several tandem repeats of 210 nucleotides within the central portion of the molecule as well as a periodic structure defined by cysteine residues. The 6699 nucleotide open reading frame of the C gene does not contain any identifiable tandem repeats or internal similarity but maintains a periodicity based on the cysteine residue spacing. The deduced amino acid sequences encoded by the two genes are most similar within the 600 amino-terminal and 600 carboxyl-terminal amino acid residues, the central portions show only limited sequence similarity. We conclude that internal repeats are not a conserved feature of variable surface proteins in Paramecium and discuss the possible importance of the regular pattern of cysteine residues.     

Opsin is a phospholipid flippase

Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ<10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.     

The immobilization antigen of Paramecium aurelia is a single polypeptide chain.

The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethaneol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (similar to 250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.     

The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite.