Figure 2.
Nub and Cub test fusions. (A) Nub (residues 1–36 of Ub) was fused to the N terminus of either a transmembrane protein (constructs 1–6) or a cytosolic protein (construct 7). The N termini of all proteins are located in the cytosol. The orientations and the numbers of membrane-spanning regions (shaded boxes) were derived from the published studies of these proteins, except for Ste14p, for which the exact number of the domains and the localization of the C terminus are not yet known. The Nub fusions 1–5 are located in the membrane of the ER; the Nub fusion 6 resides in the membrane of the early Golgi. The Nub fusion 2 is a Sec62p derivative lacking the C-terminal 60 residues. The Nub fusion 7 contains the full-length triosephosphate isomerase (Nub-Tpi1p). (B) Cub fusions. The Cub fusions 8 and 9 contain the signal sequence of the prepro-α-factor (shaded boxes), followed by either 37 (construct 8) or 65 residues (construct 9) of the mature α-factor sequence (striped boxes) and a 7-residue linker sequence (not shown). Cub fusions 10–13 contain the signal sequence of the Suc2p invertase (dark boxes) followed by 23 (construct 10), 33 (construct 11), 59 (construct 12), or 518 residues (construct 13) of the mature sequence of invertase (open boxes) and a 7-residue linker sequence (not shown). The Cub fusion 14 contains the complete sequence of S. cerevisiae triosephosphate isomerase (Tpi1p) followed by a 17-residue linker peptide and Cub. The Cub fusions 15 and 16 are the signal sequence-lacking counterparts of the fusions 10 and 12. Cub is always followed by a reporter protein. The reporter is DHFR-ha or Ura3p for the Cub fusions 8–13, and DHFR-ha for the Cub fusions 14, 15, and 16.