Fig. 1. Representation of the constructs used in this study. (A) Schematic representation of the basic TA reporter construct, GFP-17. An enhanced version of GFP (oval) is attached at its C-terminus to a linker (filled thin rectangle, L), consisting of the myc epitope followed by a repeated Gly-Ser sequence. This is attached to the entire tail region of rat b(5), which contains the TMD, flanked upstream and downstream by polar sequences (UPS and DPS, respectively). The regions between the indicated unique restriction sites in the cDNA can be easily substituted with paired synthetic oligonucleotides. The expected topology of the construct after insertion into membranes is also indicated. (B) Amino acid sequence (one-letter code) of the Linker and the UPS region present in all the constructs listed in panel (C); within the linker the residues of the myc epitope are in italics. Also shown are the sequences of the myc linker, present in the myc constructs, and the UPS linker, present in a construct in which b(5)'s UPS was deleted (these constructs are not listed in panel C — see Text). (C) Sequences of the TMD and DPS region of GFP TA constructs. The first six constructs have TMDs derived from b(5) in which amino acids have been deleted, inserted or substituted. The TMD of all these constructs is followed by the DPS of b(5). The numbers 14 through 25 in the construct names indicate the length of the TMD as predicted by hydrophilicity analysis with the scale of Engelman et al. (Engelman et al., 1986) over a window of seven residues. The residues that are predicted to be in a hydrophobic environment are shown in boldface. The construct GFP-17 contains the wild-type tail region of b(5). In the construct GFP-17-HH, the number of residues was not changed, but four substitutions (underlined) increase the TMD hydrophobicity (this results in the inclusion of one more residue in the hydrophobic region, as predicted by hydrophilicity analysis). In GFP-ST, the TMD of sialyl transferase replaces the one of b(5). In GFP-22-Nglyc, the DPS of b(5) is connected, via the couple SR, to the N-terminal sequence of bovine opsin, which contains two N-glycosylation consensus sites. The site that is predicted to be sufficiently distant from the bilayer to be used is boxed. In GFP-22-MutNglyc, both N-glycosylation sites of the preceding construct have been eliminated by AsnGln substitution (boldface). In GFP-Syn3 and Syn4, b(5)'s UPS connects to the last 29 residues of rat syntaxin 3 and 4, respectively. For all constructs, the numbers in italics enclosed by parentheses in the left column indicate the hydrophobicity of the TMD region, calculated by using the value of the scale of Engelman et al. (Engelman et al., 1986) for each residue in the sequence between the last upstream charged amino acid and the Arg residue in the DPS or for GFP-Syn 3 and 4, between the last upstream charged amino acid and the C-terminal residue.

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