Table 2.

Effects of TMD sequences on Pep12p localization

TMD	Sequence	Vacuolar locationa	ER retention signal?b
PEP12	R VYLLIVLLVMLLFIFLIM K	Out	No
Sso1	R CWLIVFAIIVVVVVVVVVPAVV K	Out	No
Nyv1	K NITLLTFTIILFVSAAFMFFYLW	Out	No
Vti1	K FISYAIIAVLILLILLVLFS K	Out	No
Tlg1	D CCIGLLIVVLIVLLVLAFIA	Out	No
Snc1	K MCLALVIIILLVVIIVPIAV H	Out	No
Cps1	R AFISGIVALIIIGTFFLTSGL H	In	(No)
Sec12	K FFTNFILIVLLSYILQFSY K	In	Yes
Ufe1	K LTTYGAIIMGVFILFL D	In	Yes
Ufe1 mutC	K -LL------------- D	In	Yes
Ufe1 mutD	K ----LL---------- D	In	Yes
Ufe1 mutE	K --------L------- D	In	Yes
Ufe1 mutF	K -LL-----L------- D	In	Yes
Ufe1 mutG	K ----LLL-L------- D	In	Yes
Ufe1 mutH	K ---------L--VVV-  D	In	Yes
Ufe1 mutI	K -LLLLL---L------ D	In/out	No
Ufe1/18	K ---------------- VL D	In/out	Yes
Ufe1/20	K ---------------- VLVL D	In/out	No
Ufe1/22	K ---------------- VLVLVL D	Out	No
Ufe1/24	K ---------------- VLVLVLVL D	Out	No

^a Vacuolar staining was classified when the indicated TMD sequences were fused to GFP–Pep12p. For a vacuolar membrane designation (out), at least 80% of the large vacuoles showed brighter fluorescence at the rim than in the center. Internal (in) means at least 95% of large vacuoles showed brighter fluorescence in the center, with no visible rims. Where neither criterion was met, the designation was in/out; see Figure 10 for examples of these. 

^b ER retention depends on the cytoplasmic domain to which the TMD is attached; none of the sequences retains Pep12p in the ER. The first six sequences were tested on Snc1p (Lewis et al., 2000; this work); the Sec12p TMD is well characterized (Sato et al., 1996); the Ufe1 mutants were tested on the Golgi SNARE Sft1p, and the elongated Ufe1 sequences were tested on Ufe1p itself (Rayner and Pelham, 1997). The Cps1p sequence has not been tested on a cytoplasmic domain known to be capable of ER retention, but Cps1p itself is not an ER protein.