Silencing p24 or p23 impairs transport of endogenous GPI-anchored proteins but not transferrin receptor. HeLa cells were transfected with siRNAs against p23 (23), p24 (24) or p25 (25). After 3 days, the cells were pulsed with [³⁵S]methionine-cysteine for 10 minutes, chased for 30 minutes (A), 90 minutes (B) or 40 minutes (C) and subjected to immunoprecipitation with antibodies against the indicated proteins. Immunoprecipitates were treated with (+) or without (−) endo-H and separated by SDS-PAGE followed by autoradiography (left panels). Quantification of endo-H resistance is given in the right panels (means ± s.d., n=3 independent experiments). *Statistically significant difference from untransfected cells (P<0.05; Student's t-test). Molecular masses in kDa are indicated on the left; un, untransfected; ctl, transfection with control siRNA.

CD59 interacts with p24 and p23. HeLa cells were either untransfected (−) or transfected (+) with Myc-p23 (A,B,E), Myc-p23 and CD59-GFP (C), HA-p24 (D,F), HA-p24 and CD59-GFP (D), as indicated. After 48 hours the cells were subjected to immunoprecipitation (Ip), SDS-PAGE, and western blotting (Wb) using the indicated antibodies. (E,F) Cells were pulsed with [³⁵S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc-p23 and HA-p24 pulled down newly synthesized endogenous CD59 and vice versa. *Bands that probably correspond to endogenous p24 and p23. IgGH, heavy chain of immunoglobulins; IgGL, light chain of immunoglobulins. Positions of molecular mass markers are indicated on the left.

GFP fused to the GPI anchor of folate receptor (GFP-GPI) interacts with p24 and p23. HeLa cells were either untransfected (−) or transfected (+) with GFP-GPI and Myc-p23 (A), or GFP-GPI and HA-p24 (B,C). (A,B) After 48 hours, the cells were subjected to immunoprecipitation (Ip) and western blotting (Wb) using the indicated antibodies. (C) Cells were pulsed with [³⁵S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc-p23 and HA-p24 pulled down newly synthesized GFP-GPI and vice versa. Positions of molecular mass markers are indicated on the left.

Sec24 isoform selectivity of p24 and p23. (A-H) HeLa cells were analyzed by double immunofluorescence microscopy using antibodies against p24 and ERGIC-53 3 days after transfection with control siRNA (control), or siRNAs against Sec24A and B, Sec24C and D, and Sec24A, B, C and D. Scale bar: 10 μm. Insets show higher magnifications of boxed regions. (I,J) Cells were transfected with Myc-p23 or HA-p24 and 48 hours later subjected to immunoprecipitation (Ip) with anti-Myc or anti-HA followed by immunoblotting with antibodies against Sec24A (A), Sec24B (B), Sec24C (C) or Sec24D (D) (upper panels). Molecular masses of the Sec24A-D isoforms (in kDa) are 113, 137, 121 or 110, respectively. Lower panels: blots with anti-Myc or anti-HA of the samples shown in the upper panel.

Co-partitioning of GPI-anchored proteins and p24-p23 into lipid rafts. HeLa cells were untransfected (A) or were transfected with Myc-p23 (B) or with HA-p24 (C). Cells were lysed in MBS-Triton X-100 at 4°C and analyzed by sucrose gradient centrifugation (see Materials and Methods). Fractions were collected and separated by SDS-PAGE followed by western blotting with antibodies against the indicated proteins. In parallel, the lipid raft fraction (15%+20%+25% sucrose) was collected, immunoprecipitated with anti-CD59 antibody (B and C), separated by SDS-PAGE, and immunoblotted with anti-CD59 and anti-Myc antibodies (B) or with anti-CD59 and HA antibodies (C). IgGL: light chain of immunglobulins. (D) Untransfected cells [to detect endogenous CD59, transferrin receptor (TfR), ERGIC-53, and BAP-31] or cells transfected with Myc-p23 or HA-p24 were pulsed for 10 minutes with [³⁵S]methionine-cysteine and subjected to sucrose density gradient centrifugation (as in A). Fractions were immunoprecipitated with antibodies against the indicated proteins or Myc and HA, and separated by SDS-PAGE followed by autoradiography. 40+endo-H, immunoprecipitated CD59 and TfR were digested with endo-H (+endo-H) before analysis. (E) Lipid raft fractions (15+20+25) from 15-minute pulse-labeled cells were pooled, immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. Note co-immunoprecipitation with CD59. Positions of molecular mass markers (in kDa) are shown on the left.

Cholesterol depletion by MβCD disrupts the interaction of GPI-anchored proteins and p24-p23. HeLa cells either untransfected (A,C,D) or transfected with Myc-p23 or with HA-p24 (B) or with glycosylated retrieval-impaired Myc-ERGIC-53 (Myc-ERGIC-53) (C,D) were treated (+) or not (−) with 20 mM MβCD for 30 minutes at 37°C. (A) Cells were lysed in MBS-Triton X-100 at 4°C, run through 5-40% sucrose gradients, subjected to SDS-PAGE and immunoblotted with antibodies against the indicated proteins. (B) Cells were lysed in 1% Triton X-100 and immunoprecipitated with anti-CD59. The immunoprecipitates were separated by SDS-PAGE and western blotting was performed with anti-CD59, anti-Myc or anti-HA. IgGL, light chain of immunoglobulins. (C,D) Cells were pulsed with [³⁵S]methionine-cysteine for 10 minutes, chased for 30 minutes (for CD59), 40 minutes (for transferrin-receptor) or 60 minutes (for Myc-ERGIC-53), lysed in 1% Triton X-100, and subjected to immunoprecipitation with antibodies against CD59, Myc or transferrin receptor. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, transferrin receptor (TfR) and Myc-ERGIC-53 (Myc-ERGIC-53) are indicated. MβCD, methyl-β–cyclodextrin.

Effect of cholesterol depletion by MβCD on the localization of endogenous CD59, ERGIC-53, p24 and Sec24C. HeLa cells were untreated (A-D) or treated (E-H) with 20 mM MβCD for 30 minutes at 37°C and analyzed by immunofluorescence microscopy using antibodies against CD59, ERGIC-53 p24 and Sec24C. Note that CD59 and p24 localize to the ER in MβCD-treated cells, whereas ERGIC-53 does not redistribute to the ER. Scale bar: 10 μm.