Fig. 2. Dimerization by disulfide bonds is required for efficient transport. (A) GM constructs. Luminal cysteines 466 and 475 of GMA₇ (– tail) and GMA₅FF (+ tail) (also compare Fig. 1) were changed to alanines, individually or in combination. (B) Oligomer formation of GMA₇ constructs bearing cysteine substitutions. 42 hours after transfection, COS cells were labeled for 5 minutes with [³⁵S]-methionine and chased as indicated. The cells were washed and lysed in the presence of 20 mM iodoacetamide and subjected to immunoprecipitation with anti-myc. Immunoprecipitates were separated by 4-10% gradient SDS-PAGE under nonreducing conditions followed by fluorography. Monomeric (1x), dimeric (2x) and hexameric (6x) forms of GM forms are indicated by arrows at the right margin. The size of molecular weight markers is indicated at the left margin. (C) Transport of GM constructs probed by pulse-chase/endo H (Fig. 1). Black bars: GMA₇ (–) and GMA₅FF (+) constructs; grey, white and hatched bars represent values of corresponding constructs with the indicated cysteine substitutions. Results are mean±s.e.m. of at least three independent experiments.