Fig. 1. A glutamine supports the ER exit determinant phenylalanine at position – 2. (A) The GM construct used for mutagenesis (Itin et al., 1995). A coiled-coil stalk domain separates the carbohydrate recognition domain (CRD) from the transmembrane domain (TMD). The di-lysine ER retrieval signal is shown in italics. Minimal anterograde transport determinants of the cytoplasmic domain are in bold (Nufer et al., 2002) (and this study). (B) Amino acid sequence of tails in GM-based constructs. Constructs 1 and 2 have been described as GMA₇ and GMA₅FF, respectively (Kappeler et al., 1997; Nufer et al., 2002). (C) COS cells were transfected with the indicated constructs shown in B and subjected to pulse-chase/endo H analysis using [³⁵S]-methionine. Cells were pulsed for 10 minutes and lysed after a chase of 60 minutes. GM constructs were immunoprecipitated with anti-myc. Immunoprecipitates were treated with endo H, separated by SDS 7-10% PAGE and analyzed by fluorography. The upper band represents the endo H-resistant and the lower band the endo H-sensitive form of GM. (D) Quantification of fluorograms including that shown in C. White bar: GMA₇; black bar: GMA₅FF. Results are mean±s.e.m. of at least three independent experiments. * statistical significance to 2 and 4; ** statistical significance to 1 and 5 (P<0.05, Student's t-test).