Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Control of the ciliary beat by cyclic nucleotides in intact cortical sheets from Paramecium

The locomotor behavior of Paramecium depends on the ciliary beat direction and beat frequency. Changes in the ciliary beat are controlled by a signal transduction mechanism that follows changes in the membrane potential. These events take place in cilia covered with a ciliary membrane. To determine the effects of second messengers in the cilia, cortical sheets were used with intact ciliary membrane as a half-closed system in which each cilium is covered with a ciliary membrane with an opening to the cell body. Cyclic nucleotides and their derivatives applied from an opening to the cell body affected the ciliary beat. cAMP and 8-Br-cAMP increased the beat frequency and the efficiency of propulsion and acted antagonistically to the action of Ca(2+). cGMP and 8-Br-cGMP increased the efficiency of propulsion accompanying clear metachronal waves but decreased the beat frequency. These results indicate that the cyclic nucleotides affect target proteins in the ciliary axonemes surrounded by the ciliary membrane without a membrane potential and increase the efficiency of propulsion of the ciliary beat. In vitro phosphorylation of isolated ciliary axonemes in the presence of cyclic nucleotides and their derivatives revealed that the action of cAMP was correlated with the phosphorylation of 29-kDa and 65-kDa proteins and that the action of cGMP was correlated with the phosphorylation of a 42-kDa protein.     

Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.

Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Regeneration of cilia in starved Tetrahymena thermophila involves induced synthesis of ciliary proteins but not synthesis of membrane lipids.

The synthesis of ciliary-membrane phospholipids and ciliary proteins was studied after deciliation in starving Tetrahymena thermophila cells. Deciliated cells regenerated the new ciliary membrane without any induced phospholipid synthesis. The constant cell volume found during the regrowth of the cilia suggests that renewal of ciliary membranes takes place by insertion of intracellular membrane material into the cell surface. In contrast with the absence of induced phospholipid synthesis during ciliary regeneration, the synthesis of ciliary proteins was found to be induced. This enhanced synthetic activity was made possible by an increased rate of intracellular protein degradation in regenerating cells. It was found that the extent of the induced synthesis strongly depends upon the growth conditions of the cells before starvation. Furthermore, it was shown that the degree of induced protein synthesis is greater for higher-molecular-weight ciliary proteins than for lower-molecular-weight species.     

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge- membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

The ciliary membrane‐associated proteome reveals actin‐binding proteins as key components of cilia

Primary cilia are sensory, antennae‐like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane‐associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high‐stoichiometric presence of actin‐binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F‐actin causes further enrichment of the actin‐binding and actin‐related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin‐binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.     

The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.     

The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization

Both the basal body and the microtubule-based axoneme it nucleates have evolutionarily conserved subdomains crucial for cilium biogenesis, function and maintenance. Here, we focus on two conspicuous but underappreciated regions of these structures that make membrane connections. One is the basal body distal end, which includes transition fibres of largely undefined composition that link to the base of the ciliary membrane. Transition fibres seem to serve as docking sites for intraflagellar transport particles, which move proteins within the ciliary compartment and are required for cilium biogenesis and sustained function. The other is the proximal-most region of the axoneme, termed the transition zone, which is characterized by Y-shaped linkers that span from the axoneme to the ciliary necklace on the membrane surface. The transition zone comprises a growing number of ciliopathy proteins that function as modular components of a ciliary gate. This gate, which forms early during ciliogenesis, might function in part by regulating intraflagellar transport. Together with a recently described septin ring diffusion barrier at the ciliary base, the transition fibres and transition zone deserve attention for their varied roles in forming functional ciliary compartments.     

The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone

In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow and which assembles a gate to maintain the specific ciliary proteome. Here we characterize the function of a novel centriolar satellite protein, synovial sarcoma X breakpoint–interacting protein 2 (SSX2IP), in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using small interfering RNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body. Cep290 takes a central role in gating proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, somatostatin receptor 3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome, and Rab8.     

A hierarchy of signals regulates entry of membrane proteins into the ciliary membrane domain in epithelial cells

The membrane of the primary cilium is continuous with the plasma membrane but compositionally distinct. Although some membrane proteins concentrate in the cilium, others such as podocalyxin/gp135 are excluded. We found that exclusion reflects a saturable selective retention mechanism. Podocalyxin is immobilized by its PDZ interaction motif binding to NHERF1 and thereby to the apical actin network via ERM family members. The retention signal was dominant, autonomous, and transferable to membrane proteins not normally excluded from the cilium. The NHERF1-binding domains of cystic fibrosis transmembrane conductance regulator and Csk-binding protein were also found to act as transferable retention signals. Addition of a retention signal could inhibit the ciliary localization of proteins (e.g., Smoothened) containing signals that normally facilitate concentration in the ciliary membrane. Proteins without a retention signal (e.g., green fluorescent protein-glycosylphosphatidylinositol) were found in the cilium, suggesting entry was not impeded by a diffusion barrier or lipid microdomain. Thus, a hierarchy of interactions controls the composition of the ciliary membrane, including selective retention, selective inclusion, and passive diffusion.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Structural basis for Arl3‐specific release of myristoylated ciliary cargo from UNC119

Access to the ciliary membrane for trans‐membrane or membrane‐associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP‐bound Arf subfamily proteins, the N‐terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.     

Evidence for a tubulin-containing lipid-protein structural complex in ciliary membranes

The proteins and lipids of the scallop gill ciliary membrane may be reassociated through several cycles of detergent solubilization, detergent removal, and freeze-thaw, without significant change in overall protein composition. Membrane proteins and lipids reassociate to form vesicles of uniform, discrete density classes under a variety of reassociation conditions involving detergent removal and concentration. Freed of the solubilizing detergent during equilibrium centrifugation, a protein-lipid complex equilibrates to a position on a sucrose density gradient characteristic of the original membrane density. When axonemal tubulin is solubilized by dialysis, mixed with 2:1 lecithin/cholesterol dissolved in Nonidet P-40, freed of detergent, and reconstituted by freeze-thaw, vesicles of a density essentially equal to pure lipid result. If the lipid fraction is derived through chloroform-methanol extraction of natural ciliary membranes, a moderate increase in density occurs upon reconstitution, but the protein is adsorbed and most is removed by a simple low ionic strength wash, in contrast to vesicles reconstituted from membrane proteins where even high salt extraction causes no loss of protein. The proteins of the ciliary membrane dissolve with constant composition, regardless of the type, concentration, or efficiency of detergent. Analytical ultracentrifugation demonstrates that monodisperse mixed micelles form at high detergent concentrations, but that membranes are dispersed to large sedimentable aggregates by Nonidet P-40 even at several times the critical micelle concentration, which suggests reasons for the efficacy of certain detergent for the production of ATP-reactivatable cell models. In extracts freed of detergent, structured polydisperse particles, but not membrane vesicles, are seen in negative staining; vesicles form upon concentration of the extract. Membrane tubulin is not in a form that will freely undergo electrophoresis, even in the presence of detergent above the critical micelle concentration. All chromatographic attempts to separate membrane tubulin from other membrane proteins have failed; lipid and protein are excluded together by gel filtration in the presence of high concentrations of detergent. These observations support the idea that a relatively stable lipid-protein complex exists in the ciliary membrane and that in this complex membrane tubulin is tightly associated with lipids and with a number of other proteins.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

Proteomic analysis of a membrane preparation from rat olfactory sensory cilia

The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research.     

Ichthyophthirius multifiliis Has Membrane-Associated Immobilization Antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis , immobilize the parasite in vitro. In order to identify ceil surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-l14 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1 - and 2-dimensiona! SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

Icthyophthirius multifiliis has membrane-associated immobilization antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.