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GLYCOPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS IN PARAMECIUM TETRAURELIA : POSSIBLE ROLE IN CHEMORESPONSE

CARRIE A. PAQUETTE, VILLA RAKOCHY, ALISON BUSH and JUDITH L. VAN HOUTEN^*

University of Vermont, Department of Biology, Burlington, VT 05405, USA
^* Author for correspondence (e-mail: jvanhout{at}zoo.uvm.edu )

View larger version (45K): [in a new window]   Fig. 6. Western blots to show that integral membrane proteins are not among those released by phospholipase C (PLC) or by washing the cells with salt/ethanol. (A) Positive and negative control western blots probed with anti-A and anti-B, anti-Ca2+-pump and anti-cyclic-AMP receptor antisera. m, pre-stained molecular mass markers. Lane 1, supernatant from PLC-treated pellicle; lanes 2, 4, bacterially expressed Ca2+ pump/glutathione-S-transferase (GST) fusion protein; lanes 3, 5, peptide from the N terminus of the cyclic AMP receptor/GST fusion protein expressed in bacteria. Lanes 1-3 were treated with anti-A and anti-B antisera, lane 4 with anti-Ca2+-pump antiserum and lane 5 with anti-cyclic-AMP receptor antiserum. The filled arrowhead points to surface antigen, and the open arrowhead points to 40-60 kDa proteins. The filled arrow marks the position of the Ca2+ pump/GST fusion protein and the open arrow to that of the N terminus of the cyclic AMP receptor/GST fusion protein. The proteins were separated on 12 % polyacrylamide gels. (B) Western blots of supernatants from sham- and PLC-treated pellicles developed with three different antibodies. m, prestained molecular mass markers. Lanes 1, 3, 5, sham-treatment supernatants; lanes 2, 4, 6, PLC treatment supernatants. Lanes 1 and 2 were treated with anti-Ca2+-pump (anti-cbd) antiserum, lanes 3 and 4 with anti-cyclic-AMP-receptor antiserum (cA rec) and lanes 5 and 6 with the anti-cross-reactive-determinant (anti-crd) antiserum. The filled arrowhead points to surface antigen, and the open arrowhead to 40-60 kDa proteins. The filled arrow points to where the intact Ca2+ pump from pellicle is expected to run (133 kDa). The open arrow points to 48 kDa, where the cyclic AMP receptor is expected to run. 6-12 % gradient polyacrylamide gels were used. (C) Western blots of salt/ethanol washes probed with three different antisera. Lane 1, anti-Ca2+-pump antiserum; lane 2, anti-A and anti-B antisera; lane 3, anti-cyclic-AMP-receptor antiserum. The filled arrow points to the expected molecular mass of the native Ca2+ pump, and the open arrow points to that of the native cyclic AMP receptor. Gradient gels from 6 to 12 % polyacrylamide were used.

View larger version (78K): [in a new window]   Fig. 1. Phospholipase treatment removes proteins with glycophosphatidylinositol (GPI) anchors from Paramecium pellicles. (A) Western blot probed with anti-A and anti-B antisera. Lane 1, pellet of sham-treated pellicle; lane 2, supernatant of sham-treated pellicle; lane 3, pellet of inositol-phosphate-specific phospholipase C (PLC)-treated pellicle; lane 4, supernatant of PLC-treated pellicle. The filled arrow points to surface antigens. The open arrow points to 40-60 kDa proteins. (B) Western blot probed with anti-cross-reacting-determinant (anti-CRD) antiserum, which binds to the cleaved GPI anchor. Lanes as in A. Gradient gels contained 8 % polyacrylamide.

View larger version (78K): [in a new window]   Fig. 2. Proteins removed from pellicle by phospholipase C (PLC) treatment at 37 and 0°C. (A) Western blot probed with anti-A and anti-B antisera, Lane 1, supernatant of sham-treated pellicle at 0°C; lane 2, supernatant of PLC-treated pellicle at 0°C; lane 3, supernatant of sham-treated pellicle at 37°C; lane 4, supernatant of PLC-treated pellicle at 37°C. (B) Same supernatants as in A but probed with anti-cross-reacting-determinant (anti-CRD) antiserum. The filled arrow points to surface antigens; the open arrow points to proteins of 40-60 kDa in gradient gels ranging from 6 to 12 % polyacrylamide.

View larger version (56K): [in a new window]   Fig. 3. Different phospholipase C (PLC) inhibitors affect exogenous and endogenous lipase activities. (A) Western blot probed with anti-A and anti-B antisera. Lane 1, supernatant from sham-treated pellicle; lane 2, supernatant from PLC-treated pellicle; lane 3, supernatant from phospholipase C (PLC)-treated pellicle with 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) and phenylmethanesulfonyl fluoride (PMSF); lane 4, supernatant of PLC-treated pellicle with p-chloromercuriphenylsulfonic acid (PCMPSA); lane 5, salt/ethanol wash proteins; lane 6, proteins from salt/ethanol wash in the presence of NCDC and PMSF; lane 7, proteins from salt/ethanol wash in the presence of PCMPSA. (B) Same as A but probed with anti-cross-reacting-determinant (anti-CRD) antiserum. The open arrow points to 40-60 kDa proteins. Surface antigens (>200 kDa) are best seen in lanes A2, A4, A5 and B2. Other proteins of interest are between 40 and 60 kDa. Gels were 8 % polyacrylamide.

View larger version (91K): [in a new window]   Fig. 5. Triton X-114 extraction solubilizes glycophosphatidylinositol (GPI)-anchored proteins. (A) Western blot probed with anti-A and anti-B antisera. Lane 1, supernatant from phospholipase C (PLC)-treated pellicle; lane 2, supernatant from PLC-treated pellicle in the presence of 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate and phenylmethanesulfonyl fluoride; lane 3, aqueous phase of Triton X-114 extraction from phase separation of pellicle proteins; lane 4, proteins from the detergent phase of Triton X-114 extraction and phase separation of pellicle. (B) Anti-cross-reactive-determinant developed electroblot of the same samples as in A. The filled arrow points to surface antigens, and the open arrow to 40-60 kDa proteins. 8 % polyacrylamide gels were used.

View larger version (106K): [in a new window]   Fig. 4. Phase-contrast micrographs of cells treated with salt/ethanol wash. (A) Typical cell treated with Dryl's solution before fixation and microscopy. (B) Typical cell treated with salt/ethanol before fixation and microscopy showing no loss of cilia. Scale bar, 10µm.

View larger version (30K): [in a new window]   Fig. 7. Western blot of salt/ethanol wash proteins showing a band reactive with antiserum against a glycophosphatidylinositol (GPI)-anchored folate receptor and reactive with anti-cross-reactive-determinant antiserum. Lane 1, treated with antifolate-binding-protein antiserum. The protein band at the arrow has an approximate molecular mass of 37 kDa. Lane 2, treated with anti-cross-reactive-determinant antiserum. Note that a band at the same molecular mass, 37 kDa, is recognized. Gels contained 12% polyacrylamide.