TABLE 1.
Properties of mutants useda
| MT mutation class | Name | Mutant tyrosine | MT | ST |
|---|---|---|---|---|
| None | WT A2 | None | WT | WT |
| Null | A185 | Null | Null | |
| Trunc | MT-Ter | Trunc/null | WT | |
| Trunc | 1387T | Trunc | WT | |
| Y1F | Y250F | 250 | Mutant | WT |
| Y315F | 315 | M | WT | |
| Y322F | 322 | M | WT | |
| Y2F | Y250/315F | 250 + 315 | M | WT |
| Y250/322F | 250 + 322 | M | WT | |
| Y315/322F | 315 + 322 | M | WT | |
| Y3FM | Y250/315/322F | 250 + 315 + 322 | M | WT |
| Y4F | Y250+/315+ | 258 + 288 + 297 + 322 | M | WT |
| Y250+/322+ | 258 + 288 + 297 + 315 | M | WT | |
| Y315+/322+ | 250 + 258 + 288 + 297 | M | WT | |
| Y5F | Y250+ | 258 + 288 + 297 + 315 + 322 | M | WT |
| Y315+ | 250 + 258 + 288 + 297 + 322 | M | WT | |
| Y322+ | 250 + 258 + 288 + 297 + 315 | M | WT | |
| Y6F | Y6F | 250 + 258 + 288 + 297 + 315 + 322 | M | WT |
| Y3Fm | Y258/288/297F | 258 + 288 + 297 | M | WT |
All mutants produce a wild-type ST except for A185, which is MTnull/STnull. MT-Ter contains a termination codon at residue 197 in the MT frame. The MT protein produced is identical to ST except for its five C-terminal residues (Lys-Arg-Arg-Ser-Glu replacing Ser-Leu-Ser-Pro) and comigrates with authentic ST. In 1387T (7), MT is truncated at amino acid 384. The absence of the carboxy-terminal hydrophobic membrane insertion domain (394 to 421) results in a cytoplasmic location. MT C-terminal tyrosines (Y) were mutated to phenylalanine (F) as described in Materials and Methods. The major (M) tyrosines, Y250, Y315, and Y322, were mutated one at a time (Y1F), pairwise (Y2F), and as a triple combination (Y3FM). The minor tyrosines, Y258, Y288, and Y297, were mutated as a group (Y3Fm). All six tyrosines were mutated simultaneously (Y6F), and the major tyrosines were added back singly, resulting in three quintuple mutants (Y5F) with a single functional tyrosine, or in pairwise combinations as three quadruple (Y4F) mutants with two functional tyrosines.