Nub and Cub fusions. (A) Nub (residues 1–36 of Ub) was fused to the N terminus of either a transmembrane protein (constructs 1–11) or a cytosolic protein (constructs 12–13). The N termini of all proteins are located in the cytosol. The orientation and the numbers of the membrane-spanning domains were obtained from published studies. The orientation of the N and the C terminus of Ste14p and its subcellular localization was a subject of this study. The Nub-attached proteins of constructs 1–5 are localized in the ER (Deshaies and Schekman, 1990; Shim et al., 1991; Finke et al., 1996; Wilkinson et al., 1996; Ballensiefen et al., 1998). The localization of the Nub-attached protein of construct 6 was a subject of this study. The Nub-attached protein of construct 7 resides in the early Golgi and of construct 8 in the late Golgi/plasma membrane (Protopopov et al., 1993; Banfield et al., 1994). The Nub-attached protein of construct 9 was shown to be in the plasma membrane (Aalto et al., 1993). The Nub-attached protein of construct 10 was found in the vacuole, and the Nub-attached protein of construct 11 was found in the outer membrane of the mitochondrion (Kiebler et al., 1993; Darsow et al., 1997; Wada et al., 1997; Srivastava and Jones, 1998). (B) Cub (residues 35–76 of Ub) was linked to the C terminus of a transmembrane protein and extended at its own C terminus by a reporter protein. The C termini of all proteins are localized in the cytosol. The information on the orientation of the N- and C-termini, the numbers of the membrane-spanning domains, and the localization of the unmodified proteins were obtained from published studies except for construct 15, where the number of membrane-spanning domains is still tentative. The Cub-attached protein of construct 14 is localized in the ER, that of construct 16 is found in the plasma membrane, and that of construct 17 is localized in the outer membrane of the mitochondrion (Jund et al., 1988; Feldheim et al., 1992; Moczko et al., 1997). The reporter (R) is RUra3p for the constructs 15–17 and RUra3p or DHFRha (Dha) for construct 14.