Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Cyclohexamide treatment does not affect Golgi localization of GREG. CHOwt or CHO stably expressing GREG-Flag were seeded on coverslips and incubated for 8 hrs in the presence (bottom panels) or absence (top panels) of cyclohexamide (CH, 100μg/ml) at 37°C, fixed and processed for immunofluorescent labeling using the antibodies against Mannosidase II and M2 (Flag). After 8hrs of treatment with a high concentration of cycloheximide, GREG-Flag is still present at the Golgi complex.
• Supplemental Figure 2 - EQ repeat is required for Golgi localization of GREG. a, Schematic representation of constructs. b, CHO cells were transiently transfected with the indicated construct for 18 hrs. After treated with Cycloheximide (50 μg ml^-1) for 1 hr, cells were fixed and processed for immunofluorescence with indicated antibodies. Images shown are representative confocal sections.
• Supplemental Figure 3 - GPI anchor mediates partitioning of GREG into lipid-enriched microdomains. a, A total membrane fraction was isolated from CHO cells transfected with GREG-Flag,ハ ΔGPI-GREG, or 23TM-GREG as indicated. Partitioning of GREG mutants into detergent-resistant membranes (DRMs) was determined by incubation of total membranes in PEN buffer containing 1%TX-100 for 0.5hr at 4°C and subsequent centrifugation at 100, 000xg for 1 hr at 4°C. The pellet (P, DRMs) and the supernatant (S, detergent-soluble proteins) were analyzed for the presence of GREG proteins by SDS-PAGE and Western blot using anti-Flag antibodies and antibodies against the indicated proteins; b, The Flag-antibody recognizes multiple bands in panel a which could reflect various glycosylation forms of GREG (affected by the membrane anchor and subcellular localization) or cross reactivity. To distinguish between these two options, GREG was treated with glycanase. Panel b shows total membranes fractions from CHO cells stably expressing GREG-Flag (20μg) that were boiled in denaturing buffer at 100°C for 10min, supplemented with NP-40 to a final concentration of 1% and incubated in the presence (lane 3) or absence (lane 4) of 25U glycanase at 37°C overnight. Proteins were precipitated with chloroform/methanol and analyzed by SDS-PAGE and Western blotting using an M2 anti-Flag antibody. As a control, postnuclear supernatants (50 μg) from either wt-CHO (lane 1) or CHO cells expressing GREG-Flag (lane 2) are shown. A non-specific band recognized by the M2 antibody is observed as well (as mentioned for Fig.2c and see also (Schafer and Braun, 1995)).
• Supplemental Figure 4 - Anomaly of Golgi structure driven by GPI anchor-deficient mutants occurs in a way independent of apoptosis and microtubule integrity. a, Panel A-D, transient expression of ΔGPI-GREG in CHO cells; Panel E-H, HeLa cells treated without (E, F) or with (G, H) 50 μg ml^-1 anisomycin for 6 hrs. Cells were analyzed for Flag (A), Man II (B), DAPI (C, F & H), GRASP 65 (E), GM130 (G) by use of a Zeiss inverted fluorescence microscope equipped with a cooled CCD camera. Panel D is a merged image of panel A-C. bホハActive forms of caspase-2 are undetectable in cells expressing GREG mutant proteins. CHO cells were transiently transfected for 16 hrs with empty vector (lane 1), GREG-Flag (lane 2), 23TM-GREG (lane 3)ホハ ΔGPI-GREG (lane 4) or HeLa cells were incubated with anisomycin (as above) (lane 5). The cell lysate was analyzed by Western blotting for the presence of activated forms of caspase-2. c, Expression of ΔGPI-GREG does not affect microtubule integrity. Cells were transiently transfected with ΔGPI-GREG for 16hrs and fixed in -20C methanol (30sec) and analyzed for Man II (green) and α-tubulin (red) by confocal immunofluorescence as described in Methods. Expression of ΔGPI-GREG was confirmed by Western blotting analysis.
• Supplemental Figure 5 - Expression of ΔGPI-GREG leads to multiple morphological changes in the Golgi region. Overview of the Golgi region of Hela cells transfected with ΔGPI-GREG-Flag. (A) Labeled with anti-Flag (10 nm gold particles), (B and C) Double labeling with anti-Flag (10 nm gold particles) and the Golgi marker Giantin (15 nm gold particles, indicated by arrowheads). In (A) and (B) formation of tightly stacked, sometimes circular membranes is shown. Note in (A) the presence of clathrin-coated membranes (arrows) at what is presumable the trans-side of an altered Golgi stack. (C) In addition to membrane stacking, accumulations of vesicles containing ΔGPI-GREG are seen. Although the origin of these membranes is unclear, some of them are labeled for Giantin as well (arrowheads). Bars, 100 nm.
• Supplemental Figure 6 - Involvement of a GPI-anchored protein in maintenance of the Golgi structure. A PIG-L deficient and recomplemented CHO cell were transfected with the NAGTI-GFP chimera cDNA. As illustrated by two representative images on the left, recomplemented cells showed a normal elongated Golgi-ribbon, whereas PIG-L, GPI deficient cells, showed fragmented Golgi structures. Right panel: random images were taken for the two cell types and elongated vs. non-elongated NAGTI labeled Golgi structures were scored for each and normalized to the number of cells counted.
• Supplemental Table 1