Fig. 1. Construction and expression of UPIa and UPIb chimeras. (A) Sequence alignment of bovine UPIa and UPIb using Clustal W (Thompson et al., 1994). The TM domains are indicated by solid lines. The boundaries of TM domains were defined by Yu et al. (Yu et al., 1994). The conserved Glu residues in TM1 and TM3 are indicated by shading. The amino acids Tyr-Trp-Ser in the cytoplasmic C-terminal tail of UPIb are probably part of the TM4 domain and are printed in bold typeface. (B) Schematic representations of the tetraspanins UPIa and UPIb. Protein domains are numbered 1 through 9, and the amino acids as well as the numbers characterizing their positions in the polypeptide sequence that define each domain are indicated. The tetraspanins UPIa and UPIb both have four TM domains (domains 2, 4, 6 and 8), which in the tetraspanin literature are also referred to as TM1, TM2, TM3 and TM4, respectively. They have a small cytoplasmic loop (domain 5) linking TM2 and TM3 and two exoplasmic loops (domains 3 and 7). In both UPIa and UPIb, domain 7 is much larger than domain 3, and bears an N-linked oligosaccharide, as indicated by the square connected to the loop in the diagram. N- and C-termini of both tetraspanins are exposed at the cytoplasmic side of the membrane (domains 1 and 9). (C) Tables listing the abbreviations that describe the chimeric constructs generated by swapping in a systematic fashion homologous domains between UPIa and UPIb. The following rules are followed for naming the chimeras: the abbreviation of the tetraspanin (Ia for UPIa and Ib for UPIb) from which most of the domains of the chimeric protein is derived is indicated first. Replaced domains are indicated second and abbreviated by the letter a (UPIa) or b (UPIb) followed by the number assigned to the exchanged domain as defined in B. For example Ia/b7 is constructed by substituting the large loop of UPIa (domain 7) with the corresponding one from UPIb, while leaving the other domains unchanged. The amino acid number and the one letter code abbreviation characterizing the beginning and the end of the UPIa and UPIb derived domains of each construct are also listed. Asterisks indicate the chimeric proteins that could be expressed at the cell surface. (D) 293T cells expressing the UPIa/UPIb chimeras indicated in C were analyzed by western blotting using antibodies directed against the large loop of UPIa or UPIb. All chimeras are well expressed, although the ratio of the glycosylated (^*) to non-glycosylated forms varied considerably.