Volume 8 issue 4 April 2007
C-Terminal Prenylation of the CLN3 Membrane Glycoprotein is required for efficient endosomal sorting to Lysosomes
S Storch, S Pohl, A Quitsch, K Falley and T Braulke

Figure S1: Tunicamycin treatment inhibits N -glycosylation of CLN3. COS7 cells transiently expressing wild type CLN3 were labeled with [³⁵S] methionine in the absence (-) or presence (+) of tunicamycin (5 µg/ml) for 16 hours followed by immunoprecipitation of CLN3 from cell extracts. Immunoprecipitates from cell extracts not-treated with tunicamycin were subsequently incubated with (+) or without (-) PNGase F. The precipitates were solubilized and subjected to Tris-Tricine-PAGE followed by fluorography. Of note, the recovery of immunoprecipitated deglycosylated CLN3 synthesized in the presence of tunicamycin is several fold higher than CLN3 immunoprecipitated from labeled cells which were subsequently deglycosylated by PNGase F treatment. These data indicate the better accessibility of antigenic determinants in non- N -glycosylated CLN3 by the anti-CLN3 antibody 242.

Figure 1(.jpg)

Figure S2: Oligomerization of claudin-2 and GFP-LIMP- 1 . (A) COS7 cells were transiently transfected with constructs coding for claudin-2, permeabilized and incubated in the absence (-) or presence (+) of cross-linker BS³ (200 µM) for 30 min. Cell extracts were subjected to SDS-PAGE followed by Western blotting with polyclonal anti-claudin antibody. The positions of monomeric, dimeric and trimeric claudin are indicated. Vector transfected cells served as controls. (B) Crosslinking of COS7 cells expressing GFP-LIMP- 1 in the presence of tunicamycin (5 µg/ml) was performed as described above followed by Western blot analysis with polyclonal anti-GFP antibody. Monomeric and dimeric GFP-LIMP- 1 are indicated. The asterisk marks possible trimeric GFP-LIMP- 1 .

Figure 2(.jpg)

Figure S3: CLN3 is present in early endosomes in mouse hippocampal neurons. Mouse hippocampal neuronal cells were transiently transfected with constructs encoding CLN3 and mutant CLN3C435S, fixed, permeabilized and stained for CLN3 (green), the early endosomal marker protein EEA1 (red), synaptic vesicle marker (SV2, red) and synaptophysin (red). Dual color images indicate co-localization. The inset represents 4-fold higher magnitude image of the region marked by the white rectangle. Scale bars are 10 µM.

Figure 3(.jpg)