Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo

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European Journal of Cell Biology 88 (2009) 577–592 www.elsevier.de/ejcb

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo Christine Klo¨ppel, Alexandra Mu¨ller, Simone Marker, Martin Simon University of Kaiserslautern, School of Biology, Erwin-Schro¨dinger-Straße, D-67663 Kaiserslautern, Germany Received 16 March 2009; received in revised form 6 May 2009; accepted 11 May 2009

Abstract Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPIanchored surface proteins in vitro: using RNAi to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNAi phenotypes show strong defects in release of GPIanchored surface proteins in vivo. Moreover, these RNAi lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo. r 2009 Elsevier GmbH. All rights reserved. Keywords: GPI cleavage; Surface protein; PI-PLC; GPI-PLC; Phospholipase C

Introduction Across kingdoms, numerous proteins on the external cell membrane leaflet are tethered to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (Englund, 1993). Because these proteins have a wide range of functions (receptors, catalytic enzymes, parasitic antigens, adhesion molecules), any defects in GPI synthesis lead to drastic patterns of disease. For example hematopoietic stem cells that fail to express a Corresponding author. Tel.: +49 63 1205 3313; fax: +49 63 1205 2496.

E-mail address: [email protected] (M. Simon). 0171-9335/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2009.05.002

certain type of GPI-anchored proteins (decay accelerating factor, DAF) lead to an uncontrolled complementmediated lysis of the blood cell called paroxysmal nocturnal hemoglobinuria (PNH) (Hu et al., 2005; Rosse, 1997). In higher eukaryotes, GPI-conjugated proteins are often involved in signaling functions in mammalian cells/tissues similar to their usage in pathogenic protists, e.g. Trypanosoma, where their random switch enables the parasite’s chronic evasion of the host immune system (Ferguson, 1999). GPI anchors are enzymatically cleavable by phospholipase C (PLC) resulting in their release into the supernatant. Treatment of kidney or liver cells with

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bacterial PI-PLC has evolved into a standard test for presence of GPI-anchored proteins (Taguchi et al., 1985). The idea that this test could be more than just a scientist’s plaything has recently been verified by demonstrating the in vivo activity of an endogenous phospholipase C acting on GPI anchors. In Trypanosoma brucei, a GPI-PLC was shown to hydrolyze the GPI anchor of the variant surface glycoproteins (VSGs) (Bu¨low and Overath, 1986; Fox et al., 1986; Hereld et al., 1986). This enzyme shows great homology to bacterial PLCs (Carrington et al., 1997) and is not comparable to any other eukaryotic PLC. In yeast, endogenous PLC activity on a GPI-anchored cAMPbinding protein has been reported after application of glucose (Mu¨ller and Bandlow, 1993; Mu¨ller et al., 1996). GPI-PLC activity in higher eukaryotes has been suggested for a lot of proteins, e.g. the alkaline phosphatase in adipocytes after insulin stimulation (Movahedi and Hooper, 1997) but it has rarely been shown. Exceptions are the cleavage of human renal dipeptidase (Park et al., 2002) and recently demonstrated GPI-PLC activity in rat adipocytes, which has interestingly also been shown to affect translocation of GPI-anchored proteins (Mu¨ller et al., 2008a, b). For higher organisms, data on the molecular characterization of responsible enzymes, their activation and localization is missing (Lauc and Heffer-Lauc, 2006). This seems surprising as also infectious prions causing fatal neurodegenerative diseases in humans and cattle are GPI anchored; strong hints exist that prion release is linked to its pathogenicity (Marella et al., 2002) and that this is mediated by an endogenous GPI-specific phospholipase (Parizek et al., 2001). Thus, the importance of information on the PLCs, that are responsible for this kind of surface protein shedding, becomes rather clear. A protein, the anchor of which is cleaved by a PI-PLC, exposes the remaining part of the anchor (1,2 cyclic monophosphate). It can be recognized by specific so-called cross-reacting determinant (CRD) antibodies (Broomfield and Hooper, 1993; Zamze et al., 1988). First seen in Trypanosoma (Barbet and McGuire, 1978), this epitope is uniquely formed on GPI cleaved by a PI-PLC, which gives us a useful tool to show PLC activity in any organism. Other types of eukaryotic PLCs are well described concerning their involvement in the signal transduction pathway: PI-PLCs cleave the membrane component 1-phosphatidyl-D-myo-inositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,4,5 trisphosphate (IP3), both acting as second messengers. All eukaryotic PLCs (except for the trypanosomes) have two regions of homology, referred to as PI-PLC-X and PI-PLC-Y box. Six isoforms are known, differing in domain architecture and tissue distribution, and some of them show additional domains: the PH domain, recruiting the proteins to different membranes, and the

calcium-binding EF-hand and C2 domains. Membrane targeting mediated by PH and C2 domains lead the PLCs to one of their possible substrates, PIP2. The intracellular localization of PI-PLCs raises the question whether and how these, or any other PLCs, could potentially cleave GPI-conjugated proteins on the cell surface. It is also still unclear, whether the trypanosomal GPI-PLC is translocated to the surface (Bu¨low et al., 1989a; Gruszynski et al., 2003). The ciliated protozoan, Paramecium, covers its surface with several types of GPI-anchored proteins which build a dense coat (Capdeville, 2000; Simon and Schmidt, 2007). Next to a set of smaller surface GPIconjugated proteins (SGPs), a multi-protein family of high-molecular-weight surface antigens (SAg) is expressed in a mutually exclusive manner, meaning that only one protein type is expressed at a time (Preer, 1986). This is comparable to the antigenic systems of parasitic protists, but the expression and switching of the different GPI-anchored surface antigens in Paramecium can be triggered by the cultivation temperature or by RNAi (Sonneborn, 1943; Simon et al., 2006a). Moreover, during serotype shifting, Paramecium was shown to release large amounts of ‘‘old’’ antigens into the medium (Momayezi et al., 2004) assuming the involvement of a PLC that catalyses the cleavage of the GPI anchor. Also spontaneous release of surface antigens was reported (Wyroba, 1980). The ciliate is therefore a good subject to study GPI-anchored proteins, especially as the amount of GPI-conjugated proteins is up to 3,5% of total cell protein, which makes them easily detectable (Preer, 1986). Several studies demonstrated that in the classical protocols for surface protein isolation, an endogenous lipase of Paramecium is activated that (i) releases the CRD epitope of the extracted proteins and (ii) can be inhibited by p-chloromercuriphenylsulfonic-acid (PCMPSA) (Benwakrim et al., 1998; Capdeville et al., 1987, Paquette et al., 2001) (for review see (Plattner et al., 2009)). As PCMPSA was shown to be a quite efficient inhibitor of PLCs essential for cleavage of GPIs (Stanton et al., 2002), the endogenous lipase is likely to be a PLC. The present study envisages the PLCs of Paramecium tetraurelia that have not been identified so far and tries to show those that are responsible for GPI cleavage in vitro and in vivo. We show that Paramecium PLCs exhibit the characteristics of higher eukaryotic PI-PLCs and that they are able to accept GPI as a substrate. Two of the PLCs we describe show distinct features concerning their catalytic domain; their RNAi phenotypes show effects both on release of GPI-anchored proteins, as well as on their transport. We anticipate that this study will generate interest in this type of newly described class of PLCs concerning the general understanding of PLC-mediated release of GPI-anchored proteins in eukaryotes.

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Materials and methods Cell culture Paramecium tetraurelia, strain d4-2 expressing serotype 51A and 51D at 30 1C and 27 1C, respectively, was cultured in wheatgrass powder (WGP) infusion medium bacterized with Klebsiella pneumoniae, supplemented with 0.8 mg/l b-sitosterol as described (Simon et al., 2006b).

Sequence and phylogenetic analysis and mRNA quantification Amino acid sequences of PLCs were aligned with ClustalW, and the set of sequences was reconstructed with the Gblocks tool (0.91b) with default settings (Dereeper et al., 2008) resulting in a 116-aa data set. The neighbor joining tree of the catalytic amino acids of the PI-PLC-X domain was calculated with MEGA4 (Tamura et al., 2007), based on p-distances and after pairwise deletion of gaps with 1000 bootstrap replicates. For mRNA quantification, integrity-checked RNA was reverse transcribed using an oligo-dT primer. Real-time PCR was carried out with the QuantiTectTM SYBRs Green kit (Qiagen, Hilden, Germany). GAPDH expression level was checked for each sample and found to reveal constant Ct values (18.9970.39).Wild-type PLC expression levels were set in relation to GAPDH and the individual knockdown of PLCs in RNAi cultures was set in relation to a culture undergoing silencing of an unrelated gene (GFP). Quantitative data from PLC genes was calculated according to Pfaffl (2001). Introns of all PLC genes were verified by cloning and sequencing of full-length RT-PCR products of iProof polymerase (Bio-Rad, Hercules, CA).

RNAi by feeding Target sequences of the respective genes were cloned into the L4440 vector: Position 1016-1412 of PLC1 (GSPATT00008002001), 658-1196 of PLC2 (GSPATT00034681001), 1453-2394 of PLC3 (GSPATT00031253001) and 1267-1648 of PLC6 (GSPATT00030070001). Note that PLC3 and PLC6 share 78 and 118 potential siRNAs (23mers) with the respective paralogous PLC5 (GSPATT00029973001) and PLC4 (GSPATT00031342001) leading to co-silencing of both paralogs (for sequence data see ParameciumDB http:// paramecium.cgm.cnrs-gif.fr/). The feeding plasmid corresponding to the 51A surface antigen gene contains position 380-874 of the open reading frame. The feeding technique was performed by transformation of plasmids into Escherichia coli HT115DE3 and IPTG induction of dsRNA synthesis as described (Galvani and Sperling, 2002). Double

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feeding experiments were carried out with mixes of bacterized media with adjusted optical density. Silencing efficiency was determined by real-time PCR to show (i) target gene knockdown, (ii) paralog knockdown and (iii) knockdown specificity, meaning that no other PLC was affected by the dsRNA.

Serotype shifts Cultures expressing serotype 51A were used for serotype shifting experiments at 30 1C with an average division rate of four divisions per day. PLC silencing was established by feeding the bacterial PLC silencing clones for three days. Then, a mixture of two thirds of PLC bacteria and one third of bacteria expressing dsRNA corresponding to the 51A gene was used to trigger the serotype shift under PLC-silencing conditions. Samples were taken 10, 13, 16, 18 and 24 h after feeding this mixture. Induced bacteria (in the same dilutions as described above) containing the empty vector (L4440) served as a negative control. Samples for immobilization with polyclonal anti-51A serum (1:200 for 20 min) contained a minimum of 50 cells and six cultures of the respective PLC (and double PLC silencing) were analyzed.

Immunofluorescence microscopy Indirect staining of surface proteins was carried out as described (Simon et al., 2006a). Paramecia were washed twice in VolvicTM and fixed in freshly prepared formaldehyde solution (4% paraformaldehyde (m/v) depolymerized by solution in phosphate-buffered saline, PBS: 0.137 M NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7,4). After washing in 50 mM glycine-PBS, blocking 3% BSA, primary antibodies (1:700), and after washing, secondary antibodies (1:400) were supplied. Stained cells were mounted into Vectashields (Vector; Burlingame, CA) to image them by microscopy using individual filters for the two fluorochromes and in a combined filter set (double filter). The primary antibodies used were the mouse Y4 monoclonal antibody (kindly provided by Y. Capdeville) and rabbit anti-51D polyclonal serum. According to this, the chosen secondary antibodies were FITC antimouse IgG and TexasReds (TR) anti-rabbit IgG (both from Sigma, Deisenhofen, Germany). Detection of the GFP-fusion protein was carried out after fixing cells in formaldehyde (8% paraformaldehyde (m/v), 1% Triton X-100 (v/v) in PBS) followed by the procedure described above with anti-GFP monoclonal mouse antibody (Roche, Mannheim, Germany) and secondary FITClabeled anti-mouse antibodies (Sigma).

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Surface protein isolation Cells (200,000) were used for salt/alcohol washes according to (Preer et al., 1981): cells were pelleted and resuspended in 400 ml VolvicTM water. Then 240 ml salt/ ethanol solution (10 mM Na2HPO4, 150 mM NaCl, 30% ethanol) was added and the tube was set on ice for 1 h. For inhibitor studies, PCMPSA (Sigma) was supplied in water at a final concentration of 1 mM, according to (Paquette et al., 2001). Cells were softly pelleted by centrifugation at 500 g to avoid contamination of intracellular proteins. Remaining cells in the supernatant were then removed by centrifugation at 10,000 g. Proteins were precipitated with 1 volume of acetone and resuspended in 150 ml 20 mM Tris-HCl, 1 mM EDTA, 25 mM KCl, 50 mM sucrose, pH 7.4, according to (Yano et al., 2003). Surface protein isolation by membrane solubilization was carried out according to (Azzouz et al., 1990). Using 200,000 cells, 3 volumes of 1.3% Triton X-100 in 50 mM Tris-HCl, pH 7.5, supplemented with completeTM protease inhibitor cocktail (Roche) were added to 1 volume of pelleted cells. After extraction for 1 h on ice, the supernatant was handled as described above for salt/ ethanol washes.

SDS-PAGE and Western blotting Gradient SDS-polyacrylamide gels (5-18%) with 3% stacking gels were used in a Biometra MaxiGel apparatus. Proteins were electro-blotted to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) in 15.6 mM Tris-HCl, 120 mM glycine, 1% SDS, 20% methanol. After staining in 0.1% Ponceau S, membranes were blocked in 5% dried milk in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.6) and probed with primary sera and secondary antibodies conjugated to alkaline phosphatase (Promega, Mannheim, Germany): primary sera were the anti-51A polyclonal serum, anti-51D polyclonal serum (kind gift of J. Forney), anti-GFP monoclonal mouse antibody (Roche, Mannheim, Germany) and anti-cross-reactive determinant (CRD) antibody (GlycoSystems, Oxford, UK; kind gift of J. van Houten, Burlington, VT). The latter antibody reacts with the carbohydrate epitope of GPIs (1,2-cyclic monophosphate) which is uniquely found in PLC-cleaved GPIanchored proteins (Zamze et al., 1988).

Results The Paramecium genome reveals six PLC candidates Screening of ParameciumDB (Arnaiz et al., 2007) resulted in the identification of six genes encoding

proteins with characteristics of eukaryotic PLCs with a prominent NH2-PLCX-PLCY-C2-COOH architecture (see Fig. 1). PLC3 and 5 exhibit an additional EF-hand domain enabling a calcium-dependent conformational change. With a molecular size of 96.2 kDa they are the largest ones (Table 1). An enzyme comparable to the GPI-PLC of Trypanosoma with a single PLCX box was not identified in the Paramecium genome. Two pairs of sequences (PLC4 and 6; PLC3 and 5) showed extremely high degrees of homology and were identified for paralogous genes of the last whole genome duplication (Aury et al., 2006). By real-time PCR we found all six genes expressed. The group of PLC2, 4 and 6 showed the highest mRNA levels, which might indicate a high turnover. Intron verification by cDNA sequencing revealed that all introns are correctly spliced with the exception of the second intron of PLC5 showing inefficient splicing (Table 1). The intron has one substitution to the conserved stereotypic flanks that has been reported for Paramecium introns (Russel et al., 1994; Jaillon et al., 2008), and loss of splicing leads to a disruption of the open reading frame because this intron is 26 nt long. As PLC5 also reveals a drastic lower mRNA level compared to all other PLCs, this gene shows characteristics of a non-functional pseudogene. Paramecium PLCs fit to PI-PLCs of higher eukaryotes by domain architecture and amino acids of the catalytic domains. In spite of the high degree of similarity, we cannot group them into any one of the isoenzyme subfamilies. Within the Paramecium PLCs, alignments of the catalytic domain show differences (Figs. 1 and 2A), and two groups become obvious: PLCs 1, 3,

Fig. 1. Domain architecture. The domain architecture shows the classical feature of eukaryotic PLCs, i.e., the catalytically active PI-PLC-X and -Y domains in an NH2-X-Y-COOH orientation, and also a C2 domain for calcium-dependent membrane translocation. PLC3 and 5 show additional EFhand domains for Ca2+-dependent conformational change. The ruler shows the protein size in amino acids. Paralogs are indicated by brackets.

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GSPATT00008002001 GSPATT00034681001 GSPATT00031253001 GSPATT00029973001 GSPATT00031342001 GSPATT00030070001 PLC1 PLC2 PLC3 PLC5 PLC4 PLC6

Two pairs of paralogs exist in PLC4+PLC6 and PLC3+PLC5. a Intron PLC5.2 was inefficiently spliced (ca. 50% unspliced mRNA) b Wild-type mRNA levels, determined by real-time PCR, were set in relation to the level of GAPDH; values reflect GAPDH  1000)/PLC. c RNAi knockdown levels of the individual PLCs were determined by real-time PCR by the difference of Ct values from PLC-silenced cultures to cultures undergoing silencing of a control gene (GFP). Values show percentage remaining transcripts of a 100% wt level.

– – – 95.9 – 94.2 47.4 88.1 44.2 94.2 44.2 100 90.7 83.1 96.2 96.2 83.0 82.6 773 709 824 825 705 705 3.7670.39 5.6470.09 1.0170.25 0.170.04 2.670.79 18.4870.55 2 1 2 2a 1 1 2370 2154 2526 2529 2146 2146

Introns Scaffold (strand) Accession number PLC

Molecular characteristics of Paramecium tetraurelia PLCs. Table 1.

20 (for) 146 (for) 122 (for) 114 (for) 122 (rev) 114 (rev)

Length (aa) Length (bp)

mRNA 1eve1b

Protein DNA

Size (kDa)

Identity (%) ref. to PLC6

Identity (%) ref. to paralog

RNAi knockdown levelc (%)

6.8470.58 24.370.2 11.3171 53.8570.23 18.270.2 10.1470.3

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5 in one and PLCs 2, 4, 6 in the other group. The latter group misses several conservative amino acids, including Ser87 that is present in all other PLCs. However, no amino acid, which has been experimentally shown to be necessary for catalytic activity on PIP2 is missing (Ellis et al., 1998). On the other hand, the alignment shows that hardly any replacement of an amino acid of these conservative amino acids resulted in a loss of enzymatic activity. As the PLC 2, 4, 6 group misses several of these amino acids, it remains questionable whether these PLCs are able to accept PIP2 for substrate or whether they are catalytically active. Compared to this group of Paramecium PLCs (PLCs 2, 4, 6), the inactive mouse PLC (PLClike2) that is involved in surface transport of G-protein subunits (Mizokami et al., 2007) shows even fewer substitutions of these conservative amino acids. Conversely, the domains of PLCs 3 and 5 are obviously intact; PLC1 shows less conservation only in the Cterminal part of the domain. As any data on PLC activity and their preferred substrate is currently unavailable, we will refer to Paramecium phospholipases as PLCs without any implication on a preferred substrate. The distinction of two groups also becomes obvious when analyzing the C2 domain (Fig. 2B): PLCs 1, 3 and 5 miss calcium-binding aspartic acid with the exception of Asp38. However, none of our PLCs shows an aspartic acid at position 86, which was shown to be necessary for membrane translocation in PLCd isoforms (Ananthanarayanan et al., 2002). As in the Paramecium enzymes we cannot find any other C2 domains exhibiting Asp86, the ciliate seems to have different requirements for membrane translocation. Calcium-dependent membrane targeting of a PIP2 cleaving PI-PLC is a step of substrate binding and therefore our analysis of C2 domains speaks for a different substrate preference of the two groups of PLCs. This conclusion would be compatible with our results obtained from the analysis of the catalytic domain. Another potential conclusion would be that PLC 1, 3 and 5 may work in a Ca2+- independent manner since they miss four of five aspartic acids. The evolutionary relationship (Fig. 3) shows clustering of the PLC 2, 4, 6 group with putative PLCs of parasitic protists. Unfortunately, experimental data are missing for these proteins. PLC3 and 5 fit to PLCs of Tetrahymena; therefore, only PLC1 seems to be unique. However, the protist PLCs are clustering together with those of higher eukaryotes and are distinct from the trypanosomal (and bacterial) PLCs. Alignments and phylogenetic analysis also do not reflect a connection of any PLC to the inactive mouse PLC-like enzyme (Mizokami et al., 2007). Moreover, a large distance of the VSG lipase (GPI-PLC) of Trypanosoma becomes obvious for all PLCs of higher organisms.

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Fig. 2. Alignment of catalytic PI-PLC-X (A) and C2 (B) domains. Invariant amino acids of mammalian PIP2cleaving PLCs are indicated below by letters. Residues indicated by (X) were shown by site-directed mutagenesis of PLCd1 to be essential for catalysis (PIP2 for substrate) (Ellis et al., 1998). In general, the ciliate PLCs group together with the mammalian ones and are not comparable to those of Trypanosoma and bacteria (not included on the alignment). PLC2, 4 and 6 show deviations in the highly conservative blocks of amino acids as they obviously miss a large number of the amino acids that are invariant in other PLCs (indicated by arrows), thus forming a separate group of enzymes. Ca2+-binding aspartatic acid residues are indicated by (m) and are absent in PLCs1, 3 and 5. Aspartatic acid86, that was shown to be necessary for Ca2+-dependent membrane translocation in PLCd isoforms (Ananthanarayanan et al., 2002), is missing in all PLCs. All amino acids are counted from the beginning of the respective domain. See legend to Fig. 3 for abbreviations of species names.

PLCs are the enzymes acting in salt/ethanol induced release of surface proteins Studies on the release of GPI-anchored surface proteins were carried out by salt/ethanol washes of cells, as this procedure was described to activate the endogenous Paramecium PLC (Benwakrim et al., 1998; Paquette et al., 2001). This was done (i) with cultures exposed to the PLC inhibitor PCMPSA and (ii) with cultures undergoing PLC silencing. The surface of Paramecium contains several classes of GPI-anchored proteins (Capdeville, 2000) and antisera specific for the large surface antigens detect several more surface proteins (Paquette et al., 2001). Therefore, surface protein isolation by salt/ethanol wash is expected to release not only the large surface antigens but also smaller surface proteins. Fig. 4A shows the effect of PCMPSA on release of surface proteins in salt/ethanol washes: in these released

protein fractions the large surface antigen (51A) and a smaller protein of 78 kDa were lacking. A protein band in the range of 55 kDa showed a strong reduction, and weaker effects of the inhibitor were obvious for release of 80-kDa and 50-kDa proteins. No effect of PCMPSA was seen for extraction of the 70-kDa protein. This was also true for release of surface antigen 51D by cultures expressing serotype 51D. However, release of other proteins (e.g. the 78-kDa protein) was inhibited in these cultures. Probing the same blot with anti-CRD antibodies showed the presence of this epitope, speaking for PLC activity, and we can exclude the activity of another kind of lipase. PCMPSA therefore inhibits cleavage of only some special types of GPIs, but not of all. Testing the specifity of the used antisera (Fig. 4B) we can indeed show that the anti-CRD antibody specifically detects proteins released by salt/alcohol extraction. Surface proteins with intact GPI anchors, isolated by membrane solubilisation are not detected, confirming

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Fig. 3. Evolutionary relationship of Paramecium PLCs with other PLCs. The neighbor joining tree (with 100 bootstrap replicates) is based on a multiple sequence alignment of the PLC catalytic domain. Beside the Paramecium tetraurelia PLCs (PLC1-6), the tree includes PLC domain-coding sequences of the following organisms: Plasmodium vivax (Pv putative PLClike, A5K7G0), Plasmodium falciparum (Pf putative PLClike, Q8IJR0), Toxoplasma gondii (Tg PLCdelta1, Q5MG88), Tetrahymena thermophila (put.PLC 1 and 2, EAR85196 and EAS04565), Danio rerio (PLCdelta3, A5D6R3), Homo sapiens (PLCdelta3, Q8N3E9), Mus musculus (PLCdelta3, Q8K2J0; PLClike2-inactive, Q8K394), Trypanosoma cruzi (Tcc VSG-Lipase, P09194), Trypanosoma brucei (Tbb VSG-Lipase, O15886). Bootstrap support values for the nodes and the evolutionary distances are indicated in the space bars.

reports of Azzouz et al. (1990). However, the blots indicate that the anti-surface antigen sera show a stronger signal for the salt/alcohol-extracted proteins. On the one hand it may be that the procedure of membrane solubilisation does not release the large surface antigens as efficiently as salt/alcohol extraction. It is also likely that antisera, which were developed by injection of salt/alcohol-extracted surface antigens, contain a large population of antibodies comparable to the anti-CRD antibody. This would also perfectly explain why all other released surface proteins of Paramecium are detected in Western blots (Fig. 4A). To look for the PLC that is involved in GPI release, we chose the 51A protein as a substrate for the silencing experiments. Note that the RNAi construct addresses both paralogs of the subgroups mentioned above and we refer to them as PLC3 and PLC6, respectively, according to the paralog with the higher expression rate. We found adequate knockdown levels for almost all PLCs in the RNAi lines (Table 1), except for PLC5 with only 53.85%. However, this gene shows characteristics of a non-functional pseudogene, as described above.

With exception of PLC3, silencing of any PLC resulted in a reduced amount of extracted surface antigens (Fig. 5) and also of those proteins, the release of which was sensitive to PCMPSA (Fig. 4). Interestingly, the release of the 55-kDa protein is less affected in PLC2+6 double silencing than in single silencing cultures and shows a weaker signal with the anti-CRD antibody (Fig. 5). One explanation would be that other, PLC-independent, release mechanisms act simultaneously. It should be kept in mind that the PLC activity studied here is certainly artificially induced because the conditions for activation of the endogenous PLC in salt/ alcohol extractions should be far from their natural trigger and we cannot exclude that these conditions also affect substrate affinity of the PLCs.

PLCs 2 and 6 are involved in antigenic variation Any PLC involvement in surface antigen release by GPI cleavage should be detectable by induction of a serotype switch during silencing of PLCs. We therefore

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Fig. 4. Effect of PCMPSA on release of surface antigens. (A) PCMPSA inhibits the removal of specific proteins during salt/ ethanol extraction of surface proteins as demonstrated with cultures expressing serotype 51A (left panel). Antisera developed against surface antigens also recognize smaller surface proteins. The large surface antigen 51A, a 78-kDa protein (below a prominent 80-kDa band) and smaller proteins of 55 and 50 kDa are not released when PCMPSA is added. Interestingly, extraction of other surface proteins seems to be unaffected or just weaker. In cultures expressing serotype 51D, release of the 51D protein interestingly shows a weaker sensitivity to PCMPSA (right panel). Smaller protein species reacted in the same way as in the 51A cultures. The same blot was probed with anti-CRD antibody as a control. This antibody detects the remaining part of a PLC-cleaved GPI anchor and, therefore, indicates PLC activity on released proteins. (B) Specifity of the anti-51A and anti-51D antisera. Surface proteins were isolated by salt/ethanol extraction (S-e) and by membrane solubilization (Sol). The anti-CRD antibody only recognizes the proteins from salt/ethanol washes, but not the antigens isolated with the intact GPI anchor. However, also the antigen-specific sera show a higher affinity to the salt/ alcohol-extracted proteins. The 80-kDa protein detected with anti-antigen sera is shown below as a loading control. Gels were 5-18% acrylamide with 40 mg protein/lane.

triggered switching of serotype 51A to 51D by RNAi in cultures that were undergoing silencing of PLCs for three days and monitored (i) the temporal duration of the shift by immobilization tests at different times (Fig. 6) and (ii) the distribution of old and new antigen on the cells (Fig. 7). The serotype switch itself was also triggered by RNAi, as silencing of the expressed antigen induces a serotype shift. Such a shift was previously well characterized for the shift from serotype 51A to 51D (Simon et al., 2006b). It was found that this antigenic switching is indeed comparable to the temperatureinduced antigen shift concerning temporal duration and

molecular regulation (Momayezi et al., 2004). We have chosen the RNAi-induced shift here because the definite trigger is constant (application of dsRNA) resulting in temporal synchronous shifts, which is impossible to induce by varying cultivation temperature. We wanted to see, how PLC knockdown influences this antigenic switching. In a time-course analysis we found equal values for already shifted cells in control cultures and in cultures with silenced PLC1 and 3 (even in double silencing), also subjected to the shift procedure indicating that they are not involved (Fig. 6). On the other hand, during 1316 h, silencing of PLC2 or 6 showed significantly more cells still reacting to the 51A serum than control cultures, but not later, indicating that the switch was strongly delayed, but successful. This delay was stronger when PLC2 and 6 were silenced simultaneously, as the most significant difference to the controls is obvious during 16-24 h. All cultures were immobilized twice, first with 51A serum and then the surviving cells with 51D serum. The immobilization reaction in general indicates the presence of antigen 51A predominantly in a qualitative way, but the amount of antigen and antibodies determines the speed of immobilization. We noticed differences in the ‘‘immobilization behavior’’ of cells undergoing PLC2+6 silencing (18 h samples), when the 51D serum was added (unpublished observations). They needed much more time to become immobilized with 51D serum and did not show the characteristic ‘‘screwdriver’’ movement during immobilization, which would lead one to assume that less 51D antigen is present on the cells. Therefore, we fixed cells of these samples looking for the exact localization of the antigens on the cell surface by indirect immunofluorescence staining as shown in Fig. 7. The control cells showed the characteristic distribution of old antigen remaining on cilia and new antigen appearing on the cell cortex (Fig. 7c-e) as was described previously for Paramecium serotype switches (Simon et al., 2006a, b). This was also true for the cells undergoing silencing of PLC1+3 (Fig. 7g and h), but we found a completely different picture for cells with silenced PLC2+6: the remaining surface antigen A was present at the poles of the cells but was not equally distributed on cilia; they also showed a complete staining of these cilia, not only at their tips, which would be expected at that late stage of the shift (Fig. 7i-k). Please note that according to the duration of the shift, the cells of PLC2+6 silencing shown in Fig. 7 were sampled several hours later than the control and PLC1+3-silenced cells that would have already completely shifted at this time. This kind of distribution shows that silencing of PLC2 and 6 obviously interferes with the release of antigens. The localization of the old antigen at the cell poles resulted from simple dilution processes during ongoing cellular divisions, considering

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Fig. 5. Western blot of salt/alcohol-extracted proteins of cultures with silenced PLCs. Surface proteins of Paramecium cultures undergoing PLC silencing were extracted in salt/ethanol washes. All cultures expressed serotype 51A, while PLCs were silenced either individually or in combinations of PLC1+3 and PLC2+6. A culture fed with bacteria containing the empty vector under the same conditions served as a control. Before surface protein isolation all cultures were immobilized with anti-51A serum to show the presence of the 51A protein on the surface. All gel lanes were loaded with the same amount of protein (40 mg/lane) and blots were all treated the same way. All PLC-silenced cultures (PLC1, 2, 3, 6 and in combination of 1+3 and 2+6) show a drastically reduced amount of released 51A protein when compared to the control, with the exception of PLC3. The most inefficient release becomes obvious in cultures silenced in PLC1 and PLC2+6 in combination. Also the 78-kDa protein below the prominent 80-kDa band shows the same release behavior, again with the most drastic reduction in PLC1- and PLC 2+6-silenced cultures (best seen on the anti-CRD blot). The 80-kDa protein, comparable to the inhibitor studies, shows only a weaker reduction during PLC silencing. Also release of the 55-kDa protein shows sensitivity to PLC silencing, with the difference that the PLC2+6 combination here shows the lowest reduction in the blot probed with anti-51A serum. The blot on the right, probed with anti-CRD antibody, indicates that all proteins are cleaved by PLC. Pictures result from different exposure times and the gel was 8% acrylamide.

Fig. 6. Effects of PLC silencing on serotype shift duration. The serotype shift from 51A to 51D (as described by Simon et al. (2006a)) was analyzed by the immobilization reaction with anti-51A serum (percentage of un-immobilized cells on Y-axis), at the indicated times after triggering. Cultures with silenced PLC1 and 3 (also in combination) show comparable duration of the shift in relation to the control culture that was fed with bacteria containing the empty vector (L4440). Those cultures that were silenced for PLC2 and 6 show almost no decreased sensitivity to the anti-51A antibodies in the first 16 h but an abrupt switch after 24 h, when about 70% of the cells did not react to the A serum any more. Such a delayed shift became slightly more obvious in cultures where PLC2+6 were silenced simultaneously. All cultures with a delayed switch shifted indeed to serotype D and the control immobilization with anti-51D serum showed a much weaker effect in PLC2+6 silenced cultures compared to controls and PLC1+ 3-silenced cells, unless the cells become immobilized finally (unpublished observations). Means7S.D. (n ¼ 6 for each culture and time). * pr0.05; *** pr0.001 (factorial ANOVA post hoc test).

that new membrane components come to the surface in the fission region of the cell as it was recently shown for syntaxin-1 in Paramecium (Kissmehl et al., 2007). Furthermore, the cells showed quite a low signal of surface antigen 51D even on the cilia and on the

non-ciliary (‘‘somatic’’) cell membrane (compared to Fig. 7c-h). This is consistent with our finding that the control immobilizations with 51D serum showed only weak effects in these cultures (unpublished observations). In conclusion, further experimentation

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Fig. 7. Distribution of old and new surface antigen on shifting cells with silenced PLCs. (a, b) Immunofluorescence images of Paramecium serotypes 51A (a) and 51D (b) with surface antigens covering the somatic and the ciliary cell membrane. (c-f) Cells of the control feeding (wild-type antigen shift, with upcoming new serotype D on the somatic cell membrane and remaining surface antigen 51A on the cilia (samples taken at 13 h after triggering), most cells showed strong 51A signals at the oral cavity (e). The cell in (f) shows remaining surface antigen A only on the tips of the cilia. (g, h) Samples taken from the shift with silenced PLC1 and 3 (13 h) showed similar staining patterns like the wild-type cells. (i-k) In contrast, cells undergoing silencing of PLC2 and 6 showed the remaining antigen on cilia at the cell poles (arrows). Samples in (i-k) were taken at 24 h to analyze cells at comparable stages during the shift (compare with Fig. 6). All cells were treated in the same manner with monoclonal antibody against antigen 51A (secondary label: FITC, green) and polyclonal serum against antigen 51D (secondary label: TexasRed) and imaged in a double filter for both fluorochromes (except (f) which was imaged with the FITC filter only).

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is necessary to determine whether PLC2 and 6 may also have an effect on the surface transport of proteins or if that may be a secondary effect of the disturbed antigen release.

A cytosolic PLC with access to the surface The experiments described above indicate that all PLCs were able to cleave GPI anchors and that they are activated in salt/ethanol extractions. The silencing phenotypes of PLC2 and 6 also indicate that these enzymes act in the same way in vivo, raising the question of their localization. Injecting the PLC2-GFP plasmid resulted in a broad cytosolic signal, in the absence of GFP in the nucleus and contractile vacuoles; cilia and cortical structures showed no defined green signals (Fig. 8A). Also the additional staining with anti-GFP antibodies did not reveal PLC localization on the

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surface. We therefore had to consider PLC-translocation mechanisms during antigenic switching. However, confirming the results of the PLC activity in salt/ethanol extraction, we found the PLC-GFP fusion protein in salt/ethanol extractions (Fig. 8B). Confirming the results of Paquette et al. (2001) that this kind of surface protein extraction only releases a special spectrum of surface proteins, we were able to show that no cytosolic proteins are released in salt/alcohol extraction. Isolating surface proteins of cultures expressing a cytosol (pure) GFP (according to Hauser et al. (2000)) did not reveal the GFP protein in Western blots (Fig. 8C, D). Therefore, release of the PLC2-GFP protein in salt/ethanol washes seems to be mediated by the PLC indicating that there is a PLC-specific translocation or secretion mechanism by which the enzyme obtains access to its substrate on the cell surface.

Discussion

Fig. 8. Subcellular localization of GFP-tagged PLC2. Cells injected with PLC2-GFP show broad cytoplasmic signals with clear absence from the macronucleus (mac) and contractile vacuoles (cv) (A, a and b). Additional staining of Tritonpermeabilized cells with anti-GFP antibodies do not reveal signals related to cilia (c). (B) Western blots probed with antiGFP antibodies show PLC2-GFP in salt/ethanol washes. Lanes 1, 2: total protein (lane 1) and salt/ethanol extract (lane 2) of PLC2-GFP-transfected cultures; lanes 3, 4: total protein (lane 3) and salt/ethanol extract (lane 4) of wild-type cells. The anti-GFP antibody recognized the GFP fusion protein of 100 kDa (arrow) not only in the total protein extract but also in the salt/ethanol-released fraction (gel was 10% acrylamide). (C) As a control we injected cultures with the empty vector, resulting in cytoplasmic expression of GFP. GFP (arrow) was detected in the total protein extract (lane 5), but not in salt/ ethanol washes (lane 6) (Gel was 5-18% acrylamide). The same blots probed with anti-51A serum served as a loading control (D). Cytoplasmic expression of (unfused) GFP is shown in (E) according to Hauser et al., (2000).

With respect to the functions of GPI-anchored proteins, we need to learn more about their release mechanisms, since these concern protein half life as well as any functions. Since the early reports of Y. Capdeville that the large surface antigens of Paramecium are GPIanchored proteins (Capdeville et al., 1987; Capdeville and Benwakrim, 1996), distribution and release of these proteins have been considered to be controlled and catalyzed by an endogenous GPI-PLC. With increasing interest on GPI-anchored proteins, their synthesis, transport and release, this issue became of increasing interest not only for protozoologists, but also in context of mammalian diseases caused by defects of GPI synthesis and turnover. The only eukaryotic PLC described as acting on GPIs is the trypanosomal GPIPLC, which is unfortunately quite similar to bacterial PLCs and not comparable to other eukaryotic PLCs (Carrington et al., 1997). The aim of the present study was to identify the Paramecium PLC that is involved in GPI modification, to analyze these enzymes and to understand the underlying mechanisms. We isolated six candidate genes of PI-PLCs, each with the characteristics of the corresponding enzymes of higher eukaryotes, but not comparable to the bacterial and trypanosomal PLCs. Two PLCs (1 and 3) had almost intact catalytic domains, but PLC2 and 6 showed a catalytic domain which distinguishes them from other PLCs. These differences however, seem to underlie strong selective pressure, because they are identical in all three proteins, also in the paralog of PLC6. As this gene, PLC4, is expressed and found correctly spliced (in contrast to PLC5), the evolutionary integrity of these three open reading frames speaks for a special role of the PLC2, 4, 6 group. We found an evolutionary relationship to PLCs in parasites of the phylum Apicomplexa

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which also express large amounts of GPI-anchored surface proteins. Interestingly, pathogenesis of Plasmodium and Toxoplasma have been reported to rely on GPI-conjugated proteins by mediating cell adhesion but also by control of immune response (Gowda and Davidson, 1999; Lekutis et al., 2001), but no involvement of a PLC has been described so far. We were not able to find any other PLC with the same catalytic domain characteristics of PLC2, 4 and 6 in any other organisms. As this work describes the affinity of these enzymes to GPI anchors this might reflect the intraspecific specifity of the enzymes for their substrate. This appears feasible considering that Paramecium also uses a modification of the glycosylinositol-phospholipid core glycan involving mannosyl phosphate. Like the PLCs, this core modification has not been found in any other organisms (Azzouz et al., 1995).

In vitro characterization of PLC activity in salt/ ethanol washes Using salt/ethanol washes, we were able to show that all PLCs contribute to the artificially induced PLC activity that was previously described to cleave the GPIs of the surface proteins of Paramecium (Paquette et al., 2001). Direct comparison of the effect of the PLC inhibitor PCMPSA to the silencing of individual PLCs reveals the same pattern of released surface proteins, which strongly supports earlier claims of the relevance of endogenous GPI-PLCs (Capdeville et al., 1987; Capdeville and Benwakrim, 1996; Paquette et al., 2001). Since the sequence data of the responsible enzymes were now available, we tried to characterize the underlying molecular mechanisms. It is not surprising that all PLCs contribute to GPI cleavage in salt/alcohol extractions because this artificial activation of the PLCs does not reflect all details of the natural mode of activation. As PLC3 is the only one that does not have a significant effect, this may be the consequence of a different localization and/or function of this enzyme: this assumption is supported, in fact, by the sequence analysis. Apart from any differences of the catalytic and substrate-binding domains, this assay does not reflect substrate specificity of the individual PLCs, but only shows the occurrence of GPI cleavage. We also cannot exclude that substrate affinity is affected by the conditions during salt/ethanol washes. However, the RNAi phenotypes of PLC2+6 in combination resulted in the most efficient inhibition of surface antigen 51A release, which fits to the in vivo results and also to the sequence analysis. However, we cannot exclude other surface protein elimination processes that happen during salt/ethanol extractions. The 55-kDa protein shows even a brighter signal in PLC2+6 silenced cultures than in the individual silencings, but the CRD

signal of the same protein is even weak although. Also for Trypanosoma, surface coat remodeling was shown to occur, next to GPI cleavage by endoproteolysis (Bu¨low et al., 1989b). In which way endogenous proteases may contribute to surface protein release, and in which way such a mechanism competes with PLC activity is not known in Paramecium and has to be investigated. Surprisingly, our blots indicate that release of other proteins show different sensitivity to PCMPSA. Some proteins show limited release, others are released in the same amount as without inhibitor. Also the release of the 51D antigen was not altered by PCMPSA. However, all these proteins show the CRD epitope. PLC cleavage of these proteins, either during PLC silencing or in presence of the inhibitor, suggests that GPI release also depends on the accessibility and on modification of the anchor. We have striking hints for such variations in GPIs: on the one hand we see strong differences in GPI signal peptides of different proteins, e.g. between 51A and 51D (unpublished observations), and on the other hand it has been experimentally shown that the fatty acid composition of GPI anchors in Paramecium is indeed varying depending on the cultivation temperature (Benwakrim et al., 1998). This fosters the assumption that different GPI anchor modifications are used, which may entail differences in cleavability. The fact that the thiol agent PCMPSA is not inhibiting the catalysis of PLCs, but rather the substrate recognition (Stanton et al., 2002) supports this assumption. As a consequence, the 51D and the 80-kDa protein seem easily accessible for PLCs, as inhibitors acting on substrate recognition do not alter their cleavage. In support of this, the extraction of 51D protein was much more efficient, as significantly more protein was isolated compared to the 51A protein. This implies that within a population, Paramecium cells may use surface protein anchors with different affinities for PLCs. GPI release would therefore be capable of being influenced by variations of the GPI anchor.

PLC2 and 6 involved in antigenic switching As we showed by induced serotype shift in vivo, only silencing of PLC2 and 6 resulted in an effect on temporal appearance and spatial distribution of old and new surface antigens. Silencing of these two PLCs interfered with the release mechanism of surface antigens, since surface antigen 51A was present for a significantly longer period on the surface as compared to controls. These showed the (wild type) distribution previously described: cilia bearing old antigen distributed equally on the surface and, at a later time, signals remaining only on the tips of cilia (Simon et al., 2006a). PLC2+6-silenced cells showed special characteristics: First, the old antigen was detected on cilia of the cell

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poles, speaking for a dilution effect of membranes and antigens. Secondly, these cilia were completely covered with 51A antigen, not only at the tips as it would be expected at that time as it can be seen on the cilia of the control cells in Fig. 7f. Finally, we also found fewer amounts of the upcoming surface antigen 51D, where we would expect much more cilia and the cortex covered with this protein. We therefore conclude that PLC-mediated release is associated with a transport of GPI-conjugated proteins along the ciliary membrane to the ciliary tip. To date, we can only speculate how intraflagellar transport or the flagellar tip complex (FTC) may be involved in the transport. Movement of the surface proteins is reminiscent to FTC-mediated transport of particles along microtubules of the axoneme (Sloboda, 2005) and these two phenomena could be coupled by organization of the GPI-anchored proteins in lipid rafts. However, this remains speculative. Silencing of PLC1 and 3, on the other hand did not show any phenotypes in vivo, regarding GPI release. As also the sequence analysis showed two separate groups within PLCs we have first hints that PLC1 and 3 may be involved in PIP2 pathways (unpublished observations). The two groups of PLCs might therefore be split into these two basic mechanisms, which is supported by differences in catalytic domains.

PLCs affecting membrane transport and GPI synthesis We have to distinguish between two phenomena: the disturbed release of the 51A protein that is associated with the untypical pattern of the proteins on the surface, and the second effect of the delayed appearance of the 51D protein on the surface. From this the idea arises that the function of the two groups of PLCs is not strictly limited to the surface. This would also be compatible with the presence of the GFP fusion protein in the cytoplasm. From inactive PLC-like enzymes in mice, we know that these PLCs (PRIPs; PLC-related inactive proteins) are involved as bridging molecules in the surface trafficking of subunits of GABAA receptors (Mizokami et al., 2007). However, if Paramecium surface antigens used the classical secretory pathway, they would not have intracellular access to the cytosolic PLCs. We therefore had to look for other mechanisms. The secretory pathway of GPI-conjugated proteins includes the addition of the anchor in the ER, transport via vesicles to the Golgi and finally to the cell surface. Here, the protein remains attached to the outer leaflet of the cell membrane (Orlean and Menon, 2007). The initial steps of GPI biosynthesis happen on the cytosolic side of the ER up to GlcN-PI, which is then flipped into the ER lumen. Compartmentalization of

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GPI-conjugated proteins in the ER saves them from PLC cleavage. The GPI precursor is sensitive to cytoplasmic PLCs, as demonstrated in Leishmania by heterologous expressed GPI-PLC resulting in anchorless proteins which are secreted into the medium (MensaWilmot et al., 1994). It was shown, moreover, in Trypanosoma that the GPI-PLC can be translocated to the ER leading to a loss of intracellular GPIs (Sandesh and Mensa-Wilmot, 2006). Assuming that PLC2 and 6 are negative regulators of GPI precursors, which is supported by experiments (i) showing that they are able to cleave GPI and (ii) are in the cytosol, the silencing of these PLCs would lead to spill over of GPI-conjugated proteins. Paramecium exhibits a large variety of GPIanchored proteins in large amounts, the post-transcriptional regulation of which is still unclear, and most of the respective genes are usually not expressed (Simon and Schmidt, 2007). As a consequence, we assume that PLC silencing results in a large amount of GPIanchored surface proteins, which were not detected immunologically in our experiments. The above mentioned weak immobilizations with anti-51D serum in PLC2+6-silenced cells support this assumption, as does the weak staining of the 51D proteins. On the other hand, as all the available antisera for surface antigens did not show an effect in the immobilization reaction (data not shown), other GPI-anchored surface proteins may be affected. Next to the long time studied multigene family of high-molecular-surface antigens, several species of smaller GPI-conjugated proteins have been described also showing temperature-dependent variations (Capdeville, 2000). Nothing is known about their regulation, but they might contribute to the hypothesized effects.

Are PLCs secreted into the medium? Our study uncovered a striking conflict between the effects of the PLC silencing and the localization of this protein. GFP localization of PLC2 showed only intracellular signals in the cytosol and the above mentioned possibilities of intracellular activity would also not explain our findings. One explanation of the observed RNAi phenotypes of PLC2+6 silencing comes from the presence of the GFP fusion protein in the salt/ alcohol washes. It was clearly shown that salt/ethanol washes of surface proteins do not contain transmembrane proteins (Paquette et al., 2001) and moreover, our results indicate that cytosolic GFP is not released in salt/ ethanol washes. Therefore, release of the PLC2-GFP protein in surface protein isolations should be mediated by the PLC and we may conclude that the presence of the GFP fusion protein in salt/ethanol washes results from a secret(ory) nature of PLCs. In vivo secretion of PLCs would be a suitable explanation for their function

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in release of GPI-anchored proteins from the surface. The significantly higher transcriptional level of PLC2, 4 and 6 also speaks for a high turnover of these enzymes, which would fit to the hypothesis. However, we do not have a proof that secretion of PLCs also happens in vivo. At first glance, secretion of cytoplasmic enzymes seems conflicting with the conventional secretory pathway. However, unconventional secretory mechanisms of cytoplasmic proteins have been shown to be widely distributed in eukaryotes, especially for proteins with extracellular signaling functions (Nickel and Seedorf, 2008; Prudovsky et al., 2008). Although the detailed mechanism of translocation is still unknown, unconventional secretion is not limited to special proteins and is also targeting cytoplasmic proteins that do not localize to ER and Golgi (Hughes, 1999; Nickel, 2003). These criteria fit perfectly to PLC2. Finally, it was also shown for Tetrahymena, a close relative of Paramecium, that at least two different PLCs are partially released and present in the culture medium (Arai et al., 1986). In summary, we identified a new class of eukaryotic PLCs that affect GPI anchors. The enzymes show characteristics of those of higher eukaryotes involved in signal transduction pathways by PIP2 cleavage. This makes them interesting for the analysis of GPIconjugated proteins in all higher organisms. Hypothesizing a secretion mechanism that happens in vivo, one of the most important questions to clarify will be whether the PLCs are constantly secreted or whether there is a special trigger, e.g. during serotype transformation. Thus, by the hypothesized mechanism cells may control GPI-conjugated protein half life, which would be a quite powerful regulation mechanism for any function of these proteins. Finally, it needs to be pointed out that modifications of GPI anchors, as mentioned above, would enable cells to select which proteins to release, in spite of secreted PLCs. In view of the drastic effects of any GPI defects and referring to DAF and prions, it will be worth looking for similar mechanism in mammals.

Acknowledgements This work was supported by grant SI 1397/1-1 from the Deutsche Forschungsgemeinschaft, DFG. We thank the Plattner group (University of Konstanz) for the kind gift of the GFP fusion vector and advice. We also thank Yvonne Capdeville (CNRS, Gif-sur-Yvette) and Judy Van Houten (University of Vermont) for ideas and discussing the subject, Michael Do¨ngi and Joachim Deitmer (Universtity of Kaiserslautern) for assistance in microscopy, Britta Hartard for comments on the manuscript and Helmut Schmidt for support of this study.

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